Methods and compositions for the treatment of metabolic disorders

ABSTRACT

The present invention is directed to a novel methods and compositions for the therapeutic intervention in hyperphenylalaninemia. More specifically, the specification describes methods and compositions for treating various types of phenylketonurias using compositions comprising BH4. Combination therapies of BH4 and other therapeutic regimens are contemplated.

BACKGROUND Field

The present invention is generally directed to the therapeuticintervention of metabolic disorders, particularly those involving aminoacid metabolism. More particularly, the present invention is directed tomethods and compositions for the treatment of phenylketonuria, vasculardiseases, ischemic or inflammatory diseases, or insulin resistance, orconditions and patients that would benefit from enhancement of nitricoxide synthase activity.

Background of the Related Technology

Phenylketonuria (PKU) is an inherited metabolic disorder that was firstidentified in the 1930s. In most cases, and until the mid-1990s, it wasthought that this is a disorder of amino acid metabolism resulting froma deficiency in the liver enzyme phenylalanine hydroxylase (PAH).Deficiencies in PAH in turn result in an excess of phenylalanine (Phe)in the brain and plasma. The deficiency in PAH ultimately manifests in alack of tyrosine, which is a precursor for the neurotransmitters.

Left undetected and untreated early in the life of an infant, PKU leadsto irreversible damage of the nervous system, severe mental retardationand poor brain development. Features other than mental retardation inuntreated patients include brain calcification, light pigmentation,peculiarities of gait, stance, and sitting posture, eczema, andepilepsy. It has been reported that an infant suffers a loss of 50IQpoints within the first year of infancy and PKU is invariablyaccompanied by at least some loss of IQ. Once detected, the condition istreated by providing the infant, and later the child, with a low Phediet. In adults, the protein supplements routinely taken by classic PKUpatients may be Phe-free with the assumption that such adults willreceive sufficient quantities of Phe through the remaining diet,controlled under a strict regimen, so that the overall diet is a low Phediet. Also, pregnant women who suffer from the condition are recommendeda diet that is low in Phe to avoid the risk of impairment of thedevelopment of the fetus and congenital malformation (maternal PKUsyndrome).

In more recent years it has been shown that pathological symptoms whichmanifest from the condition of excess of Phe, collectively termedhyperphenylalaninemia (HPA), may be divided into multiple discretedisorders, which are diagnosed according to plasma Phe concentrationsand responsiveness to a cofactor for PAH. At an initial level, HPAs maybe divided into HPA caused as a result of a deficiency in the cofactor6R-L-erythro-5, 6, 7, 8, tetrahydrobiopterin (BH4; malignant PKU) andHPA resulting from a deficiency in PAH. The latter category is furthersubdivided into at least three categories depending on the plasmaconcentration of Phe in the absence of dietary or other therapeuticintervention (referred to herein as “unrestricted plasma Pheconcentration”).

Normal plasma Phe homeostasis is tightly controlled resulting in aplasma Phe concentration of 60 μmol/L±15 μmol/L. Classical PKU (OMIM No.261600) is the most severe form of PKU and it results from null orsevere mutations in PAH, which lead to unrestricted plasma Pheconcentrations greater than 1200 μmol/L when left untreated. Individualswith classical (or severe) PKU must be treated with a strict dietaryregimen that is based on a very low Phe diet in order to reduce theirPhe concentrations to a safe range. Milder forms of HPA also have beencharacterized. A less severe form of PKU is one which manifests inplasma Phe concentrations of 10-20 mg/dL (600-1200 μmol/L), and isgenerally termed “mild PKU”. This moderate form of PKU is managedthrough the use of moderate dietary restrictions, e.g., a low totalprotein diet, but otherwise not necessarily Phe-free. Finally, mild HPA,also referred to as benign or non-PKU HPA is characterized by plasma Pheconcentrations of between 180-600 μmol/L. The individuals with non-PKUHPA are not routinely treated as they are considered to have plasma Phelevels that are within the “safe” range. Nevertheless, as mentionedabove, these Phe levels are still significantly elevated in theseindividuals as compared to normal, non-PKU subjects and may presentdetrimental sequelae in at least pregnant women and very young patients.For a more detailed review of HPA resulting from PAH deficiency, thoseof skill in the art are referred to Scriver et al., 2001(Hyperphenylalaninemia: Phenylalanine Hydroxylase Deficiency, In:Scriver C R, Beaudet A L, Sly W S, Valle D, Childs B, Vogelstein B, eds.The Metabolic and Molecular Bases of Inherited Disease. 8th ed. NewYork: McGraw-Hill, 2001: 1667-1724). Guidelines indicate that forchildren with PKU, it is preferable reduce the plasma Phe to be 360-420μmol/L.

HPA also results from defects in BH4 metabolism. BH4 is an essentialcofactor of both tyrosine and tryptophan hydroxylase, the rate limitingenzymes in the biosynthesis of the neurotransmitters dopamine andserotonin. The effects of deficiencies in dopamine and serotonin arecollectively known as “atypical” or “malignant” HPA. Thus, traditionaldiagnoses of HPA have involved a determination of whether the HPA is aresult of BH4 deficiency or PAH deficiency. Typically, diagnosis of PKUis established on the basis of a persistently elevated blood Pheconcentration. Following a positive screen for elevated blood Phe(plasma Phe >120 mon; Weglage et al., J. Inherit. Metab. Dis.,25:321-322, 2002), a differential screen is performed in which it isdetermined whether the elevated Phe is a result of BH4 deficiency or PAHdeficiency. The differential diagnosis involves determining whether theelevated Phe concentration is decreased as a result of BH4administration (BH4 loading test). The BH4 loading test typicallyinvolves a one-time load of BH4 e.g., 5-20 mg/kg being administered tothe subject who is on a normal (i.e., unrestricted) diet and determiningwhether the subject experiences a decrease in Phe levels (see e.g.,Ponzone et al., Eur. J. Pediatr. 152:655-661, 1993; Weglage et al., J.Inherit. Metab. Dis., 25:321-322, 2002.)

Typically, individuals that respond to a BH4 loading test by a decreasein plasma Phe levels are diagnosed as having a defect in BH4homeostasis. However, there have been various reports of patients with aBH4 responsive type of PAH deficiency (Kure et al., J. Pediatr.135:375-378, 1999; Lassker et al., J. Inherit. Metabol. Dis. 25:65-70,2002; Linder et al., Mol. Genet. Metab. 73:104-106, 2001; Spaapen etal., Mol. Genet. and Metabolism, 78:93-99, 2003; Trefz et al., 2001).These subjects have plasma Phe levels that are typical of moderate PKU,i.e., less than 1000 mon and typically less than 600 mon. Patients thathave severe classical PKU are not responsive to typical 24 hour BH4loading tests (Ponzone et al., N. Engl. J. Med 348(17):1722-1723, 2003).

It has been suggested that individuals that are responsive to BH4 do notrequire dietary intervention, but rather should be treated with BH4.Likewise, the converse has been suggested for subjects that have beendiagnosed as non-responsive to the BH4 loading test, i.e., thesesubjects should be treated with dietary restriction and not BH4 therapy.Ponzone et al. particularly noted that individuals that have severephenylketonuria will not respond to BH4 therapy and such therapy shouldnot be used on these patients (Ponzone et al., N. Engl. J. Med348(17):1722-1723, 2003). Thus, presently there are divergenttherapeutic regimens for treatment of HPA depending on whether or notthe individual is responsive to BH4. Moreover, it has been suggestedthat very few patients will benefit from BH4 therapy. In fact, it isthought that the only individuals with a PAH-deficient form of HPA thatwill benefit from BH4 therapy are those with mild PKU. As theseindividuals will typically have Phe levels in the safe range (i.e., lessthan 600 μM), the disease state can be controlled using moderate dietaryrestriction (see Hanley, N. Engl. J. Med 348(17):1723, 2003). Thus, BH4therapy either alone, or in combination with any other therapeuticintervention, has not being considered as a viable therapeuticintervention for the vast majority of individuals with HPA.

BH4 is a biogenic amine of the naturally-occurring pterin family.Pterins are present in physiological fluids and tissues in reduced andoxidized forms, however, only the 5,6,7,8, tetrahydrobiopterin isbiologically active. This is a chiral molecule and the 6R enantiomer ofthe cofactor is known to be the biologically active enantiomer. For adetailed review of the synthesis and disorders of BH4 see Blau et al.,2001 (Disorders of tetrahydrobiopterin and related biogenic amines. In:Scriver C R, Beaudet A L, Sly W S, Valle D, Childs B, Vogelstein B, eds.The Metabolic and Molecular Bases of Inherited Disease. 8th ed. NewYork: McGraw-Hill, 2001: 1275-1776). Despite the elucidation of the roleof BH4 deficiency in HPA, treatment with BH4 has not been suggestedbecause such treatment is very expensive, as high as $30,000 per yearfor an adolescent or adult, as compared with $6,000 forphenylalanine-restricted dietary therapy (Hanley, N. Engl. J. Med348(17):1723, 2003). Another significant problem with BH4 is that thiscompound is unstable and readily undergoes aerobic oxidation at roomtemperature (Davis et al., Eur. J. Biochem., Vol 173, 345-351, 1988;U.S. Pat. No. 4,701,455) and has a shelf-life of less 8 hours at roomtemperature (Berneggar and Blau, Mol. Genet. Metabol. 77:304-313, 2002).

Thus, to date, dietary intervention is the typical therapeuticintervention used for all patients with severe classical PKU and in manypatients with moderate PKU. Such dietary intervention typically entailsrestricting the patient to foodstuff that is composed of natural foodswhich are free from, or low in, Phe. However, in addition to eliminatingPhe, such a dietary regimen eliminates many sources of other essentialamino acids, vitamins and minerals. Consequently, withoutsupplementation, such a diet provides inadequate protein, energy,vitamins and minerals to support normal growth and development. As PKUis a manifestation of a lack of tyrosine, which arises due to the lackof hydroxylation of phenylalanine, tyrosine becomes an essential aminoacid and dietary supplements for PKU must contain a tyrosine supplement.Therefore, it is common to use nutritional formulas to supplement thediets of PKU patients. Also, for babies, it is common to use infantformulas which have a low Phe content as the sole or primary foodsource.

However, dietary protein restriction is at best an ineffective way ofcontrolling PKU in many classes of patients. For example, treatment isof paramount importance during pregnancy because high Phe levels mayresult in intrauterine retardation of brain development. However, a lowprotein diet during pregnancy may result in retarded renal developmentand is thought to produce a subsequent reduction in the number ofnephrons and potentially leads to hypertension in adulthood.(D'Agostino, N. Engl. J. Med. 348(17)1723-1724, 2003).

Poor patient compliance with a protein-restricted diet also is aproblem. The Phe-free protein formulae available are bitter tastingmaking it difficult to ensure that the patient consumes sufficientquantities of the protein to maintain the required daily intakes ofprotein, amino acids, vitamins, minerals, and the like. This isparticularly a problem with older children who may require up to 70 g,dry weight, of the formulas per day. For example, Schuett, V. E.; 1990;DHHS Publication No HRS-MCH-89-5, reports that more than 40% of PKUpatients in the US of eight years or older no longer adhere to thedietary treatment. (U.S. Pat. No. 6,506,422). Many adolescent patientsfail to rigorously follow the protein-restricted diet due to fears ofpeer attitude.

Thus, there remains a need for a therapeutic medicament to replace orsupplement and alleviate the dietary restrictions under which a PKUpatient is placed. The present invention is directed to addressing sucha need.

SUMMARY OF THE INVENTION

The invention describes intervention in metabolic disorders,particularly those involving amino acid metabolism. More particularly,the present invention is directed to methods and compositions for thetreatment of subjects exhibiting elevated phenylalanine levels, forexample, subjects suffering from hyperphenylalanemia, mildphenylketonuria or classic severe phenylketonuria; and methods andcompositions for the treatment of subjects suffering from conditionsthat would benefit from enhancement of nitric oxide synthase activity;and methods and compositions for treatment of subjects suffering fromvascular diseases, ischemic or inflammatory diseases, diabetes, orinsulin resistance.

In one aspect, the invention describes methods of treating classicsevere phenylketonuria (PKU) in a subject comprising administering tothe subject a protein-restricted diet in combination with a compositioncomprising tetrahydrobiopterin (BH4) or a precursor or derivativethereof, wherein the combined administration of the protein-restricteddiet and BH4 is effective to lower the phenylalanine concentration inthe plasma of the subject as compared to the concentration in theabsence of the combined administration. In specific embodiments, thesubject is one who does not manifest a deficiency in BH4 homeostasis.The subject may be an individual that does not manifest symptoms ofL-dopa neurotransmitter deficiency.

A subject selected from treatment according to the methods of theinvention will have an elevated plasma Phe concentration, such aconcentration may be greater than 1800 μM/L in the absence of thetherapeutic. Other embodiments contemplate that has a plasmaphenylalanine concentration of greater than 1000 μM in the absence of atherapeutic regimen. In preferred embodiments, the combinedadministration methods of the invention decrease the plasmaphenylalanine concentration of the subject to less than 600 μM. Morepreferably, it is decreased to less than 500 μM. Even more preferably,the combined administration decreases the plasma phenylalanineconcentration of the subject to 360 μM±15 μM.

The BH4 is preferably administered in an amount of between about 1 mg/kgto about 30 mg/kg, more preferably between about 5 mg/kg to about 30mg/kg. The BH4 may be administered in a single daily dose or in multipledoses on a daily basis. In some embodiments, the BH4 therapy is notcontinuous, but rather BH4 is administered on a daily basis until theplasma phenylalanine concentration of the subject is decreased to lessthan 360 μM. Preferably, wherein the plasma phenylalanine concentrationof the subject is monitored on a daily basis and the BH4 is administeredwhen a 10% increase in plasma phenylalanine concentration is observed.Preferably, the BH4 being administered is a stabilized crystallized formof BH4 that has greater stability than non-crystallized stabilized BH4.More preferably, the stabilized crystallized form of BH4 comprises atleast 99.5% pure 6R BH4. Precursors such as dihydrobiopterin (BH2), andsepiapterin also may be administered. BH4 may be administered orally

The protein-restricted diet administered in the methods herein is onethat is a phenylalanine-restricted diet wherein the total phenylalanineintake of the subject is restricted to less than 600 mg per day. Inother embodiments, the protein-restricted diet is aphenylalanine-restricted diet wherein the total phenylalanine isrestricted to less than 300 mg per day. In still other embodiments, theprotein-restricted diet is one which is supplemented with amino acids,such as tyrosine, valine, isoleucine and leucine. In certainembodiments, protein-restricted diet comprises a protein supplement andthe BH4 is provided in the same composition as the protein supplement.

In specific embodiments, the subject is one which has been diagnosed ashaving a mutant phenylalanine hydroxylase (PAH). The mutant PAH maycomprise a mutation in the catalytic domain of PAH. Exemplary suchmutations include one or more mutations selected from the groupconsisting of F39L, L48S, I65T, R68S, A104D, S110C, D129G, E178G, V190A,P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G,A395P, P407S, and Y414C.

Also contemplated herein is a method for the treating a pregnant femalehaving hyperphenylalaninemia (HPA) comprising administering to thesubject a protein-restricted diet in combination with a compositioncomprising tetrahydrobiopterin (BH4) or a precursor or derivativethereof, wherein the combined administration of the protein-restricteddiet and BH4 is effective to lower the phenylalanine concentration inthe plasma of the subject as compared to the concentration in theabsence of the combined administration. In certain embodiments, thesubject has an unrestricted plasma phenylalanine concentration ofgreater than 180 μM but less than 60004. In other embodiments, thesubject has an unrestricted plasma phenylalanine concentration ofgreater than 500 μM but less than 120004. In still other embodiments,the subject has an unrestricted plasma phenylalanine concentration ofgreater than 1000 μM.

Also contemplated is a method of treating a patient having above normalconcentration of plasma phenylalanine (e.g., greater than 180 μM/L andmore preferably, greater than 360 μM/L) comprising administering to thepatient a stabilized BH4 composition in an amount effective to produce adecrease in the plasma phenylalanine concentration of the patient.Preferably, the stabilized BH4 composition is stable at room temperaturefor more than 8 hours. The patient will likely have a plasmaphenylalanine concentration greater than 180 μM prior to administrationof the BH4. More particularly, the patient has a plasma phenylalanineconcentration of between 120 μM and 200 μM. In other embodiments, thepatient has a plasma phenylalanine concentration of between 200 μM and600 μM. In still other embodiments, the patient has a plasmaphenylalanine concentration of between 600 μM and 1200 μM. Yet anotherclass of patients to be treated are those that have an unrestrictedplasma phenylalanine concentration greater than 1200 μM. In specificembodiments, the patient is an infant, more particularly, an infanthaving a plasma phenylalanine concentration greater than 1200 μM. Inother embodiments, the patient is pregnant and pregnant patient has aplasma phenylalanine concentration of between about 200 μM to about 600μM. Pregnant patients with a plasma phenylalanine concentration greaterthan 1200 μM are particularly attractive candidates for this type oftherapy, as are patient who are females of child-bearing age that arecontemplating pregnancy. In those embodiments, in which the patient hasa plasma phenylalanine concentration greater than 1200 μM, and themethod further comprises administering a protein-restricted diet to thepatient.

The invention also contemplates a method of treating a patient havingphenylketonuria, comprising administering to the patient a stabilizedBH4 composition in an amount effective to produce a decrease in theplasma phenylalanine concentration of the patient wherein the patienthas been diagnosed as unresponsive to a single-dose BH4 loading test.Preferably, the patient is unresponsive within 24 hours of the BH4 load.

Another related aspect of the invention provides a multiple dose loadingtest that involves administration of more than one dose of BH4. The datadescribed herein demonstrates that subjects who are considered“unresponsive” to a single dose BH4 loading test can respond to multipledoses of BH4 with a significant reduction in phenylalanine levels. Inone embodiment, at least two doses of BH4 which may be between about 5mg to 40 mg are administered to a subject over a time period of morethan one day, preferably 7 days.

The treatment methods according to the invention may compriseadministering between about 10 mg BH4/kg body weight to about 200 mgBH4/kg body weight. The BH4 may be administered through any routecommonly used in practice, e.g., orally, subcutaneously, sublingually,parenterally, per rectum, per and nares. The BH4 may be administereddaily or at some other interval, e.g., every alternative day or evenweekly. The BH4 is preferably administered in combination with aprotein-restricted diet, and optionally concurrently with folates,including folate precursors, folic acids, and folate derivatives.

It is contemplated that that BH4 will be administered as part of acomponent of a therapeutic protein formulation. The protein-restricteddiet may comprise a normal diet of low-protein containing foodstuff.Alternatively, the protein-restricted diet comprises an intake ofprotein formula that is phenylalanine-free protein diet, and the subjectobtains his essential amount of Phe from remaining components of a verylow protein diet. In certain embodiments, the protein-restricted diet issupplemented with non-phenylalanine containing protein supplements. Moreparticularly, the non-phenylalanine containing protein supplementscomprise tyrosine or other essential amino acids. In other embodiments,the protein supplements may also comprise folates, including folateprecursors, folic acids, and folate derivatives.

The invention contemplates methods of treating an infant havingphenylketonuria, comprising administering a stabilized BH4 compositionto the patient in an amount effective to produce a decrease in theplasma phenylalanine concentration of the infant wherein the infant isbetween 0 and 3 years of age and the infant has a plasma phenylalanineconcentration of between about 360 μM to about 4800 μM. Prior to theadministering of BH4, the infant has a phenylalanine concentration ofabout 1200 μM and the administering of BH4 decreases the plasmaphenylalanine concentration to about 1000 μM. In other embodiments,prior to the administering of BH4 the infant has a phenylalanineconcentration of about 800 μM and the administering of BH4 decreases theplasma phenylalanine concentration to about 600 μM. In still furtherembodiments, prior to the administering of BH4 the infant has aphenylalanine concentration of about 400 μM and the administering of BH4decreases the plasma phenylalanine concentration to about 300 μM. Thetherapeutic methods contemplated herein should preferably reduce theplasma phenylalanine concentration of the infant to 360±15 μM.

Also contemplated is a composition comprising a stabilized, crystallizeform of BH4 that is stable at room temperature for more than 8 hours anda pharmaceutically acceptable carrier, diluent or excipient. Thecomposition may further comprise a medical protein supplement. In otherembodiments, the BH4 composition is part of an infant formula. In stillother embodiments, the protein supplement is phenylalanine free. Theprotein supplement preferably is fortified with L-tyrosine, L-glutamine,L-carnitine at a concentration of 20 mg/100 g supplement, L-taurine at aconcentration of 40 mg/100 g supplement and selenium. It may furthercomprise the recommended daily doses of minerals, e.g., calcium,phosphorus and magnesium. The supplement further may comprise therecommended daily dose of one or more amino acids selected from thegroup consisting of L-leucine, L-proline, L-lysine acetate, L-valine,L-isoleucine, L-arginine, L-alanine, glycine, L-asparagine monohydrate,L-tryptophan, L-serine, L-threonine, L-histidine, L-methionine,L-glutamic acid, and L-aspartic acid. In addition, the supplement may befortified with the recommended daily dosage of vitamins A, D and E. Thesupplement preferably comprises a fat content that provides at least 40%of the energy of the supplement. Such a supplement may be provided inthe form of a powder supplement or in the form of a protein bar.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further illustrate aspects of the present invention. Theinvention may be better understood by reference to the drawings incombination with the detailed description of the specific embodimentspresented herein.

FIG. 1 is a powder X-ray diffraction pattern of (6R)—BH4 Form B.

FIG. 2 is a powder X-ray diffraction pattern of (6R)—BH4 Form A.

FIG. 3 is a powder X-ray diffraction pattern of (6R)—BH4 Form F.

FIG. 4 is a powder X-ray diffraction pattern of (6R)—BH4 Form J.

FIG. 5 is a powder X-ray diffraction pattern of (6R)—BH4 Form K.

FIG. 6 is a powder X-ray diffraction pattern of (6R)—BH4 Form C.

FIG. 7 is a powder X-ray diffraction pattern of (6R)—BH4 Form D.

FIG. 8 is a powder X-ray diffraction pattern of (6R)—BH4 Form E.

FIG. 9 is a powder X-ray diffraction pattern of (6R)—BH4 Form H.

FIG. 10 is a powder X-ray diffraction pattern of (6R)—BH4 Form O.

FIG. 11 is a powder X-ray diffraction pattern of (6R)—BH4 Form G.

FIG. 12 is a powder X-ray diffraction pattern of (6R)—BH4 Form I.

FIG. 13 is a powder X-ray diffraction pattern of (6R)—BH4 Form L.

FIG. 14 is a powder X-ray diffraction pattern of (6R)—BH4 Form M.

FIG. 15 is a powder X-ray diffraction pattern of (6R)—BH4 Form N.

FIG. 16 is a mean blood phenylalanine level comparison at time zero, 3days, and 7 days for multiple daily BH4 doses of 10 mg/kg/d and 20mg/kg/d.

FIG. 17 is a comparison of daily individual blood phenylalanine levelsfor 12 adults having PKU and taking 10 mg/kg/d over 7 days.

FIG. 18 is a comparison of daily individual blood phenylalanine levelsfor 12 adults having PKU and taking 20 mg/kg/d over 7 days.

FIG. 19 is a comparison of daily individual blood phenylalanine levelsfor 8 children having PKU and taking 10 mg/kg/d over 7 days.

FIG. 20 is a comparison of daily individual blood phenylalanine levelsfor 8 children having PKU and taking 20 mg/kg/d over 7 days.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Dietary intervention is the therapeutic intervention used for allpatients with severe classical PKU and in many patients with moderatePKU. However, such dietary protein restriction leads to an inadequatesupply of protein, energy, vitamins and minerals to support normalgrowth and development. Thus, dietary protein restriction is at best anineffective way of controlling the PKU in many classes of patients,especially in pregnant women and in young children, both categories ofsubjects that require elevated amounts of protein as compared to normaladult individuals. Use of dietary restriction also is hampered by poorpatient compliance with a protein-restricted diet. In October 2000, theNational Institutes of Health issued a consensus statement on PKUscreening and management in which “research on nondietary alternativesto treatment of PKU [was] strongly encouraged.” Thus, there is anart-recognized need for a therapeutic medicament to replace and/orsupplement and alleviate the dietary restrictions under which a PKUpatient is placed.

The present application for the first time describes a pharmaceuticalintervention of PKU based on the administration of a stabilized form ofBH4. The methods and compositions for producing such a stabilized BH4compositions are described in further detail in Example 2. Thestabilized BH4 compositions of the present invention comprise BH4crystals that are stable at room temperature for longer than 8 hours.The methods and compositions of the present invention contemplatepharmaceutical compositions of the stabilized BH4 alone that may bedelivered through any conventional route of administration, includingbut not limited to oral, intramuscular injection, subcutaneousinjection, intravenous injection and the like. The compositions of thepresent invention may further comprise BH4 compositions in combinationwith an antioxidant that aids in prolonging the stability of the BH4composition. In addition, discussed in greater below, the presentinvention further comprises foodstuffs that comprise BH4. For example,the invention contemplates conventional protein powder compositions suchas PHENEX, LOFENALAC, PHENYL-FREE and the like that have been modifiedby the addition of BH4.

The present invention further contemplates the therapeutic interventionof various PKU phenotypes by administration of BH4 in combination with aprotein-restricted diet. The BH4 to be administered in combination withthe diet may, but need not necessarily, be a stabilized BH4 compositiondescribed herein. Those of skill in the art are aware of methods ofproducing a BH4 composition that is unstable at room temperature and inlight. While therapies using such a composition are hindered by theinstability of the BH4 composition, its use is still contemplated incertain combination therapies where BH4 non-responsive patientssuffering from severe classical PKU are treated with a course of BH4treatment and dietary protein restriction.

Methods and compositions for effecting the treatment of metabolicdisorders, including PKU, are described in further detail herein below.

I. PATIENTS TO BE TREATED

The present invention is directed to the treatment of a variety of HPApatient populations with methods that comprise the use of stabilized BH4compositions, or unstabilized BH4 compositions, either alone or incombination with other therapeutic regimens, for managing HPA and/orPKU. In particular, it is contemplated that any type of BH4, in astabilized or other form may be used to treat that patient populationthat has phenylalanine concentrations that are low enough that dietaryintervention is not normally used (i.e., patients with mild HPA). Suchpatients that are amenable to all forms treatment with BH4 compositionsto ameliorate the effects of mild HPA, include pregnant women andinfants with serum concentrations of less than 20004. The variouspatient populations, and their different therapeutic needs, arediscussed in further detail in the present section.

Certain embodiments of the present invention are directed to treatingclassic severe PKU by administering to the subject a protein-restricteddiet in combination with a composition comprising BH4 or a precursor orderivative thereof, wherein the combined administration of theprotein-restricted diet and BH4 is effective to lower the phenylalanineconcentration in the plasma of said subject as compared to saidconcentration in the absence of said combined administration. Inaddition, the invention also contemplates treating a pregnant femalethat has HPA by administering to the female a protein-restricted diet incombination with BH4 or a precursor or derivative thereof, such that thecombined administration of the protein-restricted diet and BH4 iseffective to lower the phenylalanine concentration in the plasma of thepregnant woman as compared to such a concentration in the absence ofsaid combined administration. In specific embodiments, therapy iscontemplated for patient who manifest Phe levels greater than 420 μM

Other embodiments of the invention entail administering a stabilized BH4composition to any individual that has HPA, characterized by a plasmaPhe concentration greater than 180 μM prior to the administration of theBH4, in an amount effective to produce a decrease in such a plasma Pheconcentration of the patient. The methods of the invention also may beused in the treatment of PKU patients that that have been diagnosed asunresponsive to a BH4 loading test. The methods of the invention will beuseful in treating an infant having PKU characterized by an elevated Pheconcentrations of between greater than 300 μM/L with the stabilized BH4compositions described herein. By “infant” the present applicationrefers to a patient that is between the ages of 0 to about 36 months.

The data described herein demonstrates that subjects who are considered“unresponsive” to the single dose BH4 loading test may in fact respondto multiple doses of BH4 with a significant reduction in phenylalaninelevels. Thus, another aspect of the invention provides a multiple doseloading test that involves administration of more than one dose of BH4.Exemplary multiple dose loading tests include administration of between5 and 40 mg/kg tetrahydrobiopterin, or more preferably 10 to 20 mg/kg,over a time period of at least 1 day, or at least 2 days, or at least 3,4, 5, 6, 7, 10 or 14 days, preferably 2-14, 3-14, or 5-10 days, and mostpreferably 7 days.

The invention provides methods of using any of the tetrahydrobiopterinpolymorphs described herein, or stable pharmaceutical preparationscomprising any of such polymorphs, for treatment of conditionsassociated with elevated phenylalanine levels or decreased tyrosinelevels, which may be caused, for example, by reduced phenylalaninehydroxylase, tyrosine hydroxylase, or tryptophan hydroxylase activity.Conditions associated with elevated phenylalanine levels specificallyinclude phenylketonuria, both mild and classic, andhyperphenylalaninemia as described elsewhere herein, and exemplarypatient populations include the patient subgroups described herein aswell as any other patient exhibiting phenylalanine levels above normal.

The invention further provides methods of using any of the polymorphsdescribed herein, or stable pharmaceutical preparations comprising anyof such polymorphs, for treatment of subjects suffering from conditionsthat would benefit from enhancement of nitric oxide synthase activityand patients suffering from vascular diseases, ischemic or inflammatorydiseases, or insulin resistance. The treatment may, for examplealleviate a deficiency in nitric oxide synthase activity or may, forexample provide an increase in nitric oxide synthase activity overnormal levels. It has been suggested that a patient suffering from adeficiency in nitric oxide synthase activity would benefit fromtreatment with folates, including folate precursors, folic acids, orfolate derivatives. Thus, it is also contemplated, that compositions andmethods disclosed herein include the concurrent treatment with folates,including folate precursors, folic acids, or folate derivatives.Exemplary folates are disclosed in U.S. Pat. Nos. 6,011,040 and6,544,994, both of which are incorporated herein by reference, andinclude folic acid (pteroylmonoglutamate), dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, one or more of the folylpolyglutamates, compounds in which thepyrazine ring of the pterin moiety of folic acid or of thefolylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, or derivatives of all the preceding compounds inwhich the N-5 or N-10 positions carry one carbon units at various levelsof oxidation, or pharmaceutically compatible salts thereof, or acombination of two or more thereof. Exemplary tetrahydrofolates include5-formyl-(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5-formimino-(6S)-tetrahydrofolic acid or (6S)-tetrahydrofolicacid, and salts thereof. as is treatment with a pharmaceuticalcomposition or foodstuff that comprises both a tetrahydrobiopterinpolymorph and a folate.

Nitric oxide is constitutively produced by vascular endothelial cellswhere it plays a key physiological role in the regulation of bloodpressure and vascular tone. It has been suggested that a deficiency innitric oxide bioactivity is involved in the pathogenesis of vasculardysfunctions, including coronary artery disease, atherosclerosis of anyarteries, including coronary, carotid, cerebral, or peripheral vasculararteries, ischemia-reperfusion injury, hypertension, diabetes, diabeticvasculopathy, cardiovascular disease, peripheral vascular disease, orneurodegenerative conditions stemming from ischemia and/or inflammation,such as stroke, and that such pathogenesis includes damaged endothelium,insufficient oxygen flow to organs and tissues, elevated systemicvascular resistance (high blood pressure), vascular smooth muscleproliferation, progression of vascular stenosis (narrowing) andinflammation. Thus, treatment of any of these conditions is contemplatedaccording to methods of the invention.

It has also been suggested that the enhancement of nitric oxide synthaseactivity also results in reduction of elevated superoxide levels,increased insulin sensitivity, and reduction in vascular dysfunctionassociated with insulin resistance, as described in U.S. Pat. No.6,410,535, incorporated herein by reference. Thus, treatment of diabetes(type I or type II), hyperinsulinemia, or insulin resistance iscontemplated according to the invention. Diseases having vasculardysfunction associated with insulin resistance include those caused byinsulin resistance or aggravated by insulin resistance, or those forwhich cure is retarded by insulin resistance, such as hypertension,hyperlipidemia, arteriosclerosis, coronary vasoconstrictive angina,effort angina, cerebrovascular constrictive lesion, cerebrovascularinsufficiency, cerebral vasospasm, peripheral circulation disorder,coronary arteriorestenosis following percutaneous transluminal coronaryangioplasty (PTCA) or coronary artery bypass grafting (CABG), obesity,insulin-independent diabetes, hyperinsulinemia, lipid metabolismabnormality, coronary arteriosclerotic heart diseases or the like so faras they are associated with insulin resistance. It is contemplated thatwhen administered to patients with these diseases, BH4 can prevent ortreat these diseases by activating the functions of NOS, increasing NOproduction and suppressing the production of active oxygen species toimprove disorders of vascular endothelial cells.

A. Characteristics of Severe Classical PKU and Methods of TreatmentThereof According to the Present Invention.

As indicated herein above in the background section, severe PKUmanifests in a plasma Phe concentration greater than 1200 μM/L and maybe found to be as high as 4800 μM/L. Patients that have this disordermust be treated with a Phe-free diet in order to bring their plasma Pheconcentrations down to a level that is clinically acceptable (typically,less than 600 μM/L, and preferably less than 300 μM/L). These patientsare only able to tolerate a maximum of between 250-350 mg dietary Pheper day (Spaapen et al., Mol. Genet and Metab. 78:93-99, 2003). As such,these patients are started on a Phe-restricted formula diet between 7-10days after birth and are burdened with this dietary restriction for theremainder their lifespan. Any alleviation of the strict dietaryrestrictions that these individuals are encumbered with would bebeneficial.

The tests used for the diagnosis of individuals with classical Phe aredescribed in further detail below in Section III. These tests haverevealed that patients with classical severe PKU are non-responsive toBH4 and require a low phenylalanine diet (Lucke et al., Pediatr. Neurol.28:228-230, 2003). In the present invention however, it is contemplatedthat this class of PKU patients should be treated with BH4 in order thatthe need for a strict phenylalanine-free diet may be alleviated.

Thus, it is contemplated that the methods of the invention will entaildetermining that the patient is suffering from classical PKU bymonitoring the plasma Phe concentration of the individual. The patientis then treated by administering a combined regimen of a low proteindiet and BH4 such that there is produced at least a 25% decrease in theplasma Phe concentrations of the patient. Preferably, the method willproduce a 30% decrease in the plasma Phe concentration. Even morepreferably, the method will produce a 40%, 50%, 60%, 70%, 80%, 90% orgreater decrease in the plasma Phe concentration of the individual (forexample, where a patient with severe classical PKU has a Pheconcentration of 4800 μM/L, a 90% decrease in the Phe concentration willproduce a plasma Phe concentration of 480 μM/L, a concentration that issufficiently low to require little dietary restriction). Of course, itshould be understood that the treatment methods of the present invention(whether for treating severe classical PKU or any other HPA describedherein), should attempt to lower the plasma Phe concentrations of thepatient to levels as close to 360 μM/L±15 μM/L as possible.

In preferred embodiments the plasma Phe concentrations of the classicalPKU patient being treated is reduced from any amount of unrestrictedplasma Phe concentration that is greater than 1000 μM/L to any plasmaPhe level that is less than 600 μM/L. Of course, even if the combinedtreatment with the BH4 and the protein-restricted diet produces a lesserdecrease in plasma Phe concentration, e.g., to a level of between 800μM/L to about 1200 μM/L, this will be viewed as a clinically usefuloutcome of the therapy because patients that have a plasma Pheconcentration in this range can manage the disease by simply restrictingthe amount of protein in the diet as opposed to eating a Phe-restrictedformula, thereby resulting in a marked improvement in the quality oflife of the individual, as well as leading to greater patient compliancewith the dietary restriction.

Any increase in the amount of dietary Phe levels that can be toleratedby the patient as a result of the treatment will be considered to be atherapeutically effective outcome. For example, it is contemplated thatas a result of administering the BH4-based therapy, the patient will beable to increase his/her intake of dietary Phe from 250-350 mg/day to350-400 mg/day (i.e., the Phe tolerance phenotype of the patient isaltered from that of a classic PKU patient to a moderate PKU patient).Of course, it would be preferable that the therapeutic interventiontaught herein would allow the patient to increase his/her intake ofdietary Phe from 250-350 mg/day to 400-600 mg/day (i.e., the Phetolerance phenotype of the patient is altered from that of a classic PKUpatient to a mild PKU patient), or even more preferably, to allow thepatient to have an intake of greater than 600 mg Phe/day (i.e., normaldietary intake).

B. Characteristics of BH4-Non Responsive PKU Patients and Methods ofTreatment Thereof According to the Present Invention.

A second group of patients that can be treated with the methods of thepresent invention are those individuals that have a been determined tohave an elevated plasma Phe concentrations i.e., any concentration thatis greater than 200 μM/L, but have been diagnosed to be non-responsiveto BH4 therapy (as determined by the BH4 loading test described below).Such patients may include those individuals that have mild PKU (i.e.,plasma Phe concentrations of up to 600 μM/L), individuals that havemoderate PKU (i.e., plasma Phe concentrations of between 600 μM/L toabout 1200 μM/L), as well as patients that have classic severe PKU(i.e., plasma Phe concentrations that are greater than 1200 μM/L).

The patients that are non-responsive to BH4 therapy are given BH4 incombination with a reduced amount of protein in their diet in order todecrease the plasma Phe concentrations of the patient. The methods ofthe present invention are such that the administration of the BH4therapy produces a greater decrease in the plasma Phe concentrations ofthe patient as compared to the decrease that is produced with the samedietary protocol administered in the absence of the BH4 therapy.

In preferred embodiments, the patients are administered a compositionthat comprises a stabilized, crystallized form of BH4 characterized inExample 2 described herein below. This BH4 composition differs fromthose previously available in the art in that it is more stable at roomtemperature than the preparations previously known to those of skill inthe art, e.g., those available in the BH4 loading kits (SchircksLaboratories, Jona, Switzerland.) Thus, the BH4 formulation may bestored at either room temperature or refrigerated and retain greaterpotency than the previously available BH4 compositions. As such, it iscontemplated that this form of BH4 will have a greater therapeuticefficacy than a similar concentration the previously available BH4compositions. This greater efficacy may be used to produce atherapeutically effective outcome even in patients that were previouslyidentified as being non-responsive to BH4.

As with the subset of patients described in Section IA above, the BH4non-responsive patients described in the present section may be treatedby the stabilized BH4 compositions either alone or in combination withdietary restrictions. The dietary restrictions may be as a diet thatrestricts the Phe intake by providing a synthetic medical proteinformula that has a diminished amount of Phe or alternatively, thedietary restriction may be one which simply requires that the patientlimit his/her overall protein intake but nevertheless allows the patientto eat normal foodstuffs in limited quantities.

The preferred therapeutic outcomes discussed for classical PKU patientsin Section IA above are incorporated into the present section byreference. Preferred therapeutic outcomes for patients with moderate PKU(i.e., patients that has an unrestricted plasma Phe concentration of 600μM/L to 1200 μM/L) include at least a 25% decrease in the plasma Pheconcentrations of the patient. Preferably, the method will produce a 30%decrease in the plasma Phe concentration. Even more preferably, themethod will produce a 40%, 50%, 60%, 70%, 80%, 90% or greater decreasein the plasma Phe concentration of the individual (for example, where apatient with moderate classical PKU has a Phe concentration of 1000μM/L, a 90% decrease in the Phe concentration will produce a plasma Pheconcentration of 100 μM/L, a concentration that is sufficiently low torequire little dietary restriction).

In preferred embodiments, the plasma Phe concentrations of the moderatePKU patient being treated is reduced from any amount of unrestrictedplasma Phe concentration that is between 600 μM/L to 1200 μM/L to anyplasma Phe level that is less than 300 μM/L. A particularly preferredtreatment with the BH4 (either alone or in combination with a dietaryrestriction) produces a decrease in plasma Phe concentration, e.g., to alevel of between 200 μM/L to about 400 μM/L, which will be viewed as aclinically useful outcome of the therapy because patients that have aplasma Phe concentration in this range can manage the disease by simplyrestricting the amount of protein in the diet as opposed to eating aPhe-restricted formula. Indeed, in many studies, it is taught that suchpatients may even eat a normal diet.

Any increase in the amount of dietary Phe levels that can be toleratedby the patient as a result of the treatment will be considered to be atherapeutically effective outcome. For example, it is contemplated thatas a result of administering the BH4-based therapy (either alone or incombination with other therapeutic intervention), the patient will beable to increase his/her intake of dietary Phe from 350-400 mg/day to400-600 mg/day (i.e., the Phe tolerance phenotype of the patient isaltered from that of a moderate PKU patient to a mild PKU patient). Ofcourse, it would be preferable that the therapeutic intervention taughtherein would allow the patient to increase his/her intake of dietary Phefrom 350-400 mg/day to 400 to allow the patient to have an intake ofgreater than 600 mg Phe/day (i.e., normal dietary intake).

Even if the patient being treated is one who manifests only mild PKU,i.e., has a dietary allowance of 400-600 mg Phe intake/day) will benefitfrom the BH4-based therapies of the present invention because it isdesirable to produce a normalized plasma Phe concentration that is asclose to 360 μM/L±15 μM/L as possible. For such patients, a preferredtherapeutic outcomes will include at least a 25% decrease in the plasmaPhe concentrations of the patient. Preferably, the method will produce a30% decrease in the plasma Phe concentration. Even more preferably, themethod will produce a 40%, 50%, 60%, or greater decrease in the plasmaPhe concentration of the individual (for example, where a patient withmild PKU has a Phe concentration of 600 μM/L, a 60% decrease in the Pheconcentration will produce a plasma Phe concentration of 360 μM/L, i.e.,an acceptable, normal concentration of plasma Phe).

In preferred embodiments, the plasma Phe concentrations of the mild PKUpatient being treated is reduced from any amount of non-restrictedplasma Phe concentration that is between 400 μM/L to 600 μM/L to anyplasma Phe level that is less than 100 μM/L. Of course, even if thetreatment with the BH4 (either alone or in combination with a dietaryrestriction) produces a lesser decrease in plasma Phe concentration,e.g., to a level of between 200 μM/L to about 400 μM/L, this will beviewed as a clinically useful outcome of the therapy.

Any increase the amount of dietary Phe levels that can be tolerated bythe patient as a result of the treatment will be considered to be atherapeutically effective outcome. For example, it is contemplated thatas a result of administering the BH4-based therapy (either alone or incombination with other therapeutic intervention), the patient will beable to increase his/her intake of dietary Phe from 400-600 mg/day(i.e., the Phe tolerance phenotype of the patient is altered from thatof a mild PKU patient to a mild HPA patient) to allow the patient tohave an intake of greater than 600 mg Phe/day (i.e., normal dietaryintake).

Furthermore, even if the patient is one who only manifests the symptomsof non PKU HPA, i.e., has an elevated plasma Phe concentration of up to600 μM/L, but is otherwise allowed to eat a normal protein diet willbenefit from the BH4 therapies of the invention because it has beenshown that elevated Phe concentrations have significant effects on theIQ of such individuals. Moreover, as discussed below, BH4-basedtherapeutic intervention of subjects with special needs, e.g., pregnantwomen and infants, is particularly important even if that patient'splasma Phe levels are within the perceived “safe” level of less than 200μM/L.

C. Maternal PKU and Methods of Treatment Thereof According to thePresent Invention.

Metabolic control of plasma Phe levels in PKU women planning conceptionand those who are pregnant is important because of the seriousconsequences to the fetus exposed to even moderately elevated Phe levelsin utero, regardless of the PAH status of the fetus. Therapeutic controlof plasma Phe concentration is especially important in the firsttrimester of pregnancy, as failure to achieve adequate control willresult in disorders including microcephaly, mental deficiency andcongenital heart disease.

For example, the NIH Consensus Statement (vol 17 #3, October 2000) onPhenylketonuria reported that exposure of a fetus to maternal Phe levelsof 3-10 mg/dL produced a 24% incidence of microcephaly, whilst thoseexposed to greater than 20 mg/dL (i.e., greater than 1200 μM/L) had a73% incidence of microcephaly. Likewise congenital heart disease wasfound in over 10% of children exposed to maternal Phe levels that weregreater than 20 mg/dL. Importantly, it has been noted that levels of Phegreater than 6 mg/dL significantly decrease the IQ of the child. Thus,it is imperative to ensure that the plasma Phe concentration of womenwith all forms of phenylketonuria, even those manifesting the mildestHPA, must be tightly controlled in order to avoid the risk of maternalPKU syndrome. However, the acceptable target levels for the plasma Pheconcentrations of PKU women that have been used in U.S. clinics haveranged between 10 mg/dL and 15 mg/dL, which are much higher than the 2-6mg/dL levels recommended for pregnant women or the 1-4 mg/dL that areused in British and German clinics to diminish the risks of developingmaternal PKU syndrome.

Another important consideration for pregnant women is their overallprotein intake. During pregnancy, it is important that women eatsufficient protein because it has been suggested that a low protein dietduring pregnancy will result in retarded renal development andsubsequent reduction in the number of nephrons and potentially leads tohypertension in adulthood. (D'Agostino, N. Engl. J. Med.348(17)1723-1724, 2003). The following table provides exemplaryguidelines for the recommended total dietary protein intake for variousindividuals.

TABLE United States Guidelines for dietary protein requirementsRecommended Total Age Protein Intake (g) Infant 6 months or less 13 6months-1 year 14 1-3 years 16 Children 4-6 years 24 7-10 years 28 Males11-14 years 45 15-18 years 59 19-24 58 25-50 63 51+ 63 Females 11-14years 46 15-18 years 44 19-24 46 25-50 50 51+ 50 Pregnant 60 Lactating65

The actual amount of protein ingested depends on the Phe content of theprotein. The amino acid profiles of plant proteins is different fromanimal proteins. For example, with a focus on starches and vegetables, ageneral rule of 45-50 mg/Phe per gram of protein may suffice. However,an accepted standard for evaluating the constituents amino acids of aprotein is an egg white, which contains 3.5 grams of protein of which204 mg is Phe.

As can be seen from the above exemplary guidelines, in the UnitedStates, the recommended protein intake for women of child-bearing age(e.g., less than 51) is from about 44 to 50 g/day, whereas pregnantwomen require are recommended an intake of about 60 g/day. In Canada andthe United Kingdom, the recommended protein intake for pregnant women isin the order of about 70 g/day and 52 g/day. Thus, the need to ensurethat the plasma Phe concentration levels of pregnant women are tightlycontrolled is further complicated by the fact that this group of PKUpatient requires more protein than non-pregnant PKU females ofcomparable age.

In view of the above, it is contemplated that BH4-based therapies of thepresent invention will be particularly useful in pregnant women. It iscontemplated that a woman suffering from any form of HPA who is pregnantor is contemplating pregnancy will be placed on a course of BH4 therapyto ensure that her plasma Phe concentration levels are maintained asclose to 180 μM/L to about 360 μM/L as possible. Such a course oftherapy will preferably allow that woman to increase her level of normalprotein intake.

The discussion of levels of plasma Phe concentrations and the degrees towhich such Phe concentrations should be decreased discussed herein abovein Sections IA and IB are incorporated into the present section forpregnant women.

D. Managing PKU in Infants and Methods of Treatment Thereof According tothe Present Invention.

As discussed herein throughout, it has been determined that an elevationin the plasma Phe concentration in infants (ages zero to 3 years old)results in significant drop in IQ of the child. However, as has beendiscussed elsewhere in the specification, patients that have an elevatedplasma Phe concentration of anywhere up to 400 μM/L do not normallyreceive any dietary intervention. Thus, infants at the age of zero to 3years in age suffer from significant deleterious effects from thepresent therapies. The instant application contemplates treating anyinfant having an unrestricted plasma Phe concentration that is greaterthan 360 μM/L±150 μM/L with a therapeutic composition that comprises BH4in order to produce a beneficial decrease the plasma Phe concentrationof that subject.

In preferred embodiments, the infant is aged between zero and 3 years ofage and has an unrestricted plasma Phe concentration of about 1200 μM/Lprior to the administration of BH4 and said administration decreases theplasma Phe concentration. Preferably, the plasma Phe concentration isdecreased to from greater than 1800 to about 1500 μM/L, about 1200 μM/L,about 1100 μM/L, about 1000 μM/L, about 900 μM/L, about 800 μM/L, about700 μM/L, about 600 μM/L, about 550 μM/L, about 500 μM/L, about 450μM/L, 400 μM/L, about 350 μM/L, about 300 μM/L, about 275 μM/L, about250 μM/L upon administration. In other embodiments, the infant is agedbetween zero and 3 years of age and has an unrestricted plasma Pheconcentration of greater than 1200 μM/L and preferably, this plasma Pheconcentration is decreased to about 800 μM/L, or more preferably toabout 500 μM/L or even more preferably to about 360 μM/L uponadministration of BH4, either alone or in combination with diet. Thoseof skill in the art would understand that the invention contemplatestreating infants with unrestricted plasma Phe concentrations of greaterthan 360 μM/L with BH4 to produce decreases in such plasma Pheconcentrations. The discussion of therapeutic reductions of plasma Pheconcentrations in Sections IA and IB above are incorporated herein byreference. Further, any decrease over 10% of the initial unrestrictedplasma Phe concentration will be considered a therapeutic outcome forthe therapeutic regimens for the infants. It should be understood thatthe BH4 therapies may be combined with dietary restrictions to effectthe therapeutic decrease in plasma Phe concentrations in such infants.

II. COMPOSITIONS FOR USE IN THE TREATMENT

The present invention contemplates therapeutic intervention of PKU/HPA.Such intervention is based initially on the use of BH4. The BH4 may beused alone or in combination with dietary restrictions. Further the BH4and/or dietary restrictions may further be combined with othertherapeutic compositions that are designed, for example to combat othermanifestations of PKU, such as for example, large neutral amino acids toprevent Phe accumulation in the brain (see Koch et al., Mol. Genet.Metabol. 79:110-113, 2003) or tyrosine supplementation. The presentsection provides a discussion of the compositions that may be used inthe treatments contemplated herein.

A. BH4 Compositions

BH4 is a cofactor in Phe hydroxylation and prior to the presentinvention, it was shown that less than 2% of patients having an elevatedPhe at birth have defects in BH4 synthesis. With those individuals thatwere identified as being BH4 responsive, it was suggested that thepatients would be non-responsive to dietary intervention and hence,those individuals were fed a normal diet but given BH4 therapy alone.Thus, prior to the present invention, there was much skepticism in theart as to the therapeutic benefits of BH4 administration to PKU/HPApatients. However, as discussed herein throughout, BH4 may beadministered for a therapeutic intervention of patients that have beendiagnosed as non-BH4 responsive. Moreover, the present inventors showthat BH4 therapy can be combined with dietary restrictions to produce atherapeutic outcome in both individuals that are responsive to a BH4loading test as well as individuals that are non-responsive to BH4loading.

U.S. Pat. Nos. 5,698,408; 2,601,215; 3,505,329; 4,540,783; 4,550,109;4,587,340; 4,595,752; 4,649,197; 4,665,182; 4,701,455; 4,713,454;4,937,342; 5,037,981; 5,198,547; 5,350,851; 5,401,844; 5,698,408 andCanadian application CA 2420374 (each incorporated herein by reference)each describe methods of making dihydrobiopterins, BH4 and derivativethereof that may be used as compositions for the present invention. Anysuch methods may be used to produce BH4 compositions for use in thetherapeutic methods of the present invention.

U.S. Pat. Nos. 4,752,573; 4,758,571; 4,774,244; 4,920,122; 5,753,656;5,922,713; 5,874,433; 5,945,452; 6,274,581; 6,410,535; 6,441,038;6,544,994; and U.S. Patent Publications US 20020187958; US 20020106645;US 2002/0076782; US 20030032616 (each incorporated herein by reference)each describe methods of administering BH4 compositions for non-PKUtreatments. Each of those patents is incorporated herein by reference asproviding a general teaching of methods of administering BH4compositions known to those of skill in the art, that may be adapted forthe treatment of PKU/HPA as described herein.

In addition to the above general methods of making BH4, the presentinvention particularly contemplates making and using a BH4 compositionwhich is a stabilized BH4 composition. Preferably the stabilized BH4composition is in crystalline form. Methods of making the stabilized BH4compositions for use in the present invention are described in Example2. Such a crystalline form may prove useful as an additive toconventional protein formulas for the treatment of PKU. The crystallineform also may conveniently be formed into a tablets, powder or othersolid for oral administration. The forms and routes of administration ofBH4 are discussed in further detail in the Pharmaceutical Compositionssection below.

In preferred embodiments, it is contemplated that the methods of thepresent invention will provide to a patient in need thereof, a dailydose of between about 10 mg/kg to about 20 mg/kg of BH4. Of course, oneskilled in the art may adjust this dose up or down depending on theefficacy being achieved by the administration. The daily dose may beadministered in a single dose or alternatively may be administered inmultiple doses at conveniently spaced intervals. In exemplaryembodiments, the daily dose may be 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg, 24 mg/kg, 26mg/kg, 28 mg/kg, 30 mg/kg, 32 mg/kg, 34 mg/kg, 36 mg/kg, 38 mg/kg, 40mg/kg, 42 mg/kg, 44 mg/kg, 46 mg/kg, 48 mg/kg, 50 mg/kg, or more mg/kg.

Regardless of the amount of BH4 administered, it is desirable that theadministration decreases the plasma Phe concentration of the patients tothe concentrations discussed in Section I for the various types ofpatients.

B. Dietary Protein

In addition to administering BH4 and related analogs to HPA/PKUpatients, it is contemplated that the dietary protein of the patientsalso may be restricted or modified. Those of skill in the art are awareof various commercially available protein formulas for use in thetreatment of PKU. Such formulas include MAXIMAID, PHENEX 1, PHENEX 2(Ross Laboratories, Liverpool, UK), LOFENALAC, PHENYL-FREE(Mead-Johnson), and the like.

Those of skill in the art may use the referenced protein formulas, whichare generally free of Phe concentrations. The protein formulas often aresupplemented with amino acids that are deficient in PKU patients. Suchamino acids include, for example, L-tyrosine, and L-glutamine. It hasbeen suggested that it may be desirable to supplement the diet of PKUpatients with valine, isoleucine and leucine (see U.S. Pat. No.4,252,822). In certain clinical manifestations, the toxic effects of PKUare caused by Phe blocking the brain uptake of other amino acids such astyrosine and tryptophan. It has been found that supplementing the dietof a PKU patient with excess of such large neutral amino acids blocksPhe uptake into the brain and lowers brain Phe levels. Thus, it iscontemplated that for the methods of the present invention, the dietaryregimen may further be supplemented with compositions that comprise oneor more of these amino acids (Koch et al., Mol. Genet. Metabol.79:110-113, 2003).

Further, as it is known that L-carnitine and taurine which are normallyfound in human milk and other foodstuffs of animal origin also should besupplied in addition to the protein restriction. In certain embodiments,the L-carnitine may be supplied as 20 mg/100 g of protein supplement,and the taurine may be supplied as 40 mg/100 g protein supplement inorder to help supply amounts of these factors normally found in humanmilk and foods of animal origin.

In addition, those of skill in the art are by reference to the 2000National Academy of Sciences-National Research Council Dietary ReferenceIntakes for a further listing of other components, such as essentialvitamins and minerals that should be supplied to the patient to ensurethat other supplements are being provided despite the dietary proteinrestriction.

Referring to the Table presented in Section IC above for total proteinamounts and the figures presented in Section I in general for thedesirable plasma Phe concentrations, one of skill in the art will beable to determine the amount of dietary protein restriction that isrequired and thus adjust the diet of the patient accordingly. Taking forexample, a male of about 11-14 years of age, that individual shouldpreferably receive 45 g protein/day. In the event that the individual isone that has severe classic PKU, his unrestricted plasma Pheconcentration will likely be greater than 1200 μM/L, and most, if notall of the dietary protein source for that individual is likely to befrom a powdered protein supplement, which preferably lowers his plasmaPhe concentrations to less than 600 μM/L. By administering BH4 to thatsubject, a therapeutic outcome would be one which produces greaterdecrease in the plasma Phe concentrations of patient or alternatively,the therapeutic outcome is one in which the individual's plasma Pheconcentrations is lowered to a similar degree, but that individual isable to tolerate protein from a normal diet rather than from a dietaryformula.

Similarly, a male of about 11-14 years of age, is one who has moderatePKU, it may be possible using the methods of the present invention togive him the allotted 45 g protein/day through a normal protein intakerather than a restricted formula. Determining whether the methods of theinvention are effective will entail determining the plasma Pheconcentrations of the patient on a regular basis to ensure that theplasma Phe concentrations remain below at least 400 μM/L. Tests fordetermining such concentrations are described below. Preferably,concentrations of less than or about 360 μM/L are achieved.

III. IDENTIFYING AND MONITORING PATIENT POPULATIONS

As discussed herein throughout, it will be necessary in variousembodiments of the present invention to determine whether a givenpatient is responsive to BH4 therapy, and to determine the phenylalanineconcentrations of the patient both initially to identify the class ofPKU patient being treated and during an ongoing therapeutic regimen tomonitor the efficacy of the regimen. Exemplary such methods aredescribed herein below.

A. BH4 Loading Test

In order to identify a patient as being responsive to BH4, those ofskill in the art perform a “BH4 loading” test. Two types of loadingtests have been used to achieve the differential diagnosis of HPA. Thefirst is a simple oral BH4 loading test and the second is a combinedphenylalanine/BH4 loading test.

The simplest BH4 loading test is one in which exogenous BH4 isadministered and the effects of the administration on lowering of plasmaPhe concentrations is determined. Intravenous loading of 2 mg/kg BH4 wasinitially proposed by Danks et al., (Lancet 1:1236, 1976), as BH4 ofgreater purity has become available it has become possible to performthe test using an oral administration of BH4 in amounts of about 2.5mg/kg body weight. Ultimately, a standardized approach was proposed byNiederwieser et al. in which a 7.5 mg/kg single oral dose of BH4 isadministered (Eur. J. Pediatr. 138:441, 1982), although somelaboratories do still use upwards of 20 mg BH4/kg body weight. This testallows discrimination between patients that have HPA due to a deficit inBH4 or through a deficiency in PAH.

In order for the simple BH4 loading test to produce reliable results,the blood Phe levels of the patient need to be higher than 400 μM/L.Therefore, it is often customary for the patient to be removed from thePKU diet for 2 days prior to performing the loading test. A BH4 test kitis available and distributed by Dr. Schircks Laboratories (Jona,Switzerland). This kit recommends a dosage of 20 mg BH4/kg body weightabout 30 minutes after intake of a normal meal.

As indicated above, the Phe concentration of a patient ideally needs tobe higher than 400 μM/L in order to obtain an accurate BH4 reading. Inthe combined Phenylalanine/BH4 loading test, an oral administration ofPhe (100 mg/kg body weight) plus BH4 (20 mg/kg body weight) allowsselective screening of all BH4 deficiencies. Typically, the Phe isadministered in an oral dose and it is followed approximately one hourlater with BH4. The plasma Phe levels are monitored before and atconvenient time intervals (e.g., 1, 3, 5, 9, 13 and 25 hours) post-Pheadministration.

In either the simple BH4 loading test or the combined Phe/BH4 loadingtest, it has been suggested that a decrease in plasma Phe of more than30% of the plasma Phe value prior to BH4 challenge within 24 hourspost-load is indicative of BH4 responsiveness (Spaapen et al, Mol.Genet. and Metabol., 78:93-99, 2003).

Other methods of performing BH4 loading tests also may be used.Exemplary such tests are described in e.g., Muntau et al., (N. Engl. J.Med. 347(26):2002) and Berneggar and Blau (Mol. Genet. Metabol.77:304-313, 2002).

In Berneggar and Blau, the BH4 loading test uses 20 mg/kg BH4 and bloodsampling for phenylalanine and tyrosine is performed at 0, 4, 8, and 24hours to differentiate between BH4-responders and non-responders. Thetest us carried out after at least 3 hours of fasting. Urine samples ofneopterin and biopterin are tested before the test. After an oralapplication of 6R BH4 (20 mg·kg body weight), normal food intake isallowed during the etire testing period. Blood samples are assayed forPhe and Tyr measurements at 0, 4, 8 and 24 hours. Another urine sampleis collected between 4-8 hours. Dihydropteridine reductase activity alsomay be measured anytime during the test. In patients that have plasmaphenylalanine levels less than 400 μM/L or patients already on alow-phenylalanine diet, Berneggar and Blau recommend a combinedphenylalanine-BH4 test in which 100 mg Phe/kg body weight isadministered orally 3 hours before the BH4 administration.

Berneggar and Blau calculated BH4-responsiveness as “phenyalaninehydroxylation” at 4 and 8 hours after loading and was expressed as apercentage of the phenylalanine eliminated. The slope (S) of the graphsof “hydroxylation rates” at 0, 4 abd 8 hours are compared fro differentBH4 products and different groups of patient. The slop discriminatesbetween non-responders, slow responders and responders. The slowresponders (see FIG. 5 in Berneggar and Blau) need more time to reachthe cut-off values of 360 μM/L and that the effectiveness ofadministered BH4 depends on the initial phenylalanine levels. Theseauthors recommend that for some patients with plasma Phe of less than800 μM/L and for most patients with a plasma Phe greater than 1200 μM/L,a Phe measurement should be taken at 21 hours. A plot of Phe/S vs. timecan be used to estimate the time needed to reach the therapeutic “safe”plasma Phe values of less than 360 μM/L.

Muntau et al. (2002) also provide exemplary BH4 loading tests that canbe used to calculate the times and concentrations of BH4 administration.Again these authors employed a combined PHE/BH4 loading test in whichpatients are give a meal that contains 100 mg Phe/kg body weight. Onehour after the meal, the patients are given an oral dose of 20 mg/kg BH4(Schirks Laboratories). Blood phenylalanine concentrations aredetermined by electrospray ionization mass spectrometry before Pheloading as well as before, and at 4, 8, and 15 hours after BH4 loading.Newborns may be breast fed, whereas older patients are give astandardized protein intake (10 mg Phe/kg) between 6-8 hours after BH4loading. Muntau also describe methods for Phe oxidation. After a 4-hourfast and an overnight fast a total of 6 mg/kg 13C labeled Phe dissolvedin dextrose solution is administered orally. Breath samples are thencollected over a period of 180 minutes and stored in evacuated glasstubes. The samples are then analysed using isotope ration massspectrometry and the recovery of carbon 13 is calculated (Treacy et al.,Pediat. Res. 42:430-5, 1997)

Muntau et al. classify patients as BH4 responsive when the blood Phelevels 15 hours post-BH4 challenge have decreased by more than 30% fromthe value obtained prior to the BH4 administration. An improvement inthe rate of Phe oxidation, as determined by measurements of carbondioxide obtained during the 180 minutes of testing, was consideredsignificant when the supplementation with BH4 increased the value of Pheoxidation by at least 15%.

Those of skill in the art will be able to use any of theabove-referenced methods to determine whether an individual will beresponsiveness to BH4. However, other equivalent and related methods fordetermining BH4 responsiveness also may be known to those of skill inthe art and may be used instead of the methods described above.

B. Determination of Phe concentrations

There are numerous methods for determining the presence of Phe in blood(Shaw et al., Analytical Methods in Phenylketonuria-ClinicalBiochemistry, InBickett et al. Eds. Phenylketonuria and Some OtherInborn Errors of Amino Acid Metabolism, Stuttgart, Georg Thiem Verlag,47-561971,). Typically, phenylalanine and tyrosine concentrations aredetermined from the serum of a patient using a fluorometric assay. Thisassay relies on the formation of fluorescent substance whenphenylalanine is heated with ninhydrin in the presence of leucylalanine(McCaman et al., J. Lab. Clin. Med. 59:885-890, 1962.)

The most popular method for determining Phe concentrations is theGuthrie test in which discs are punctured from filter paper that hasbeen saturated with a blood sample from the patient. The uniform discsare incubated in a tray of agar that has been seeded with Bacillussubtilis and contains a specific inhibitor of Bacillus subtilis growth.As the phenylalanine transfers from the uniform discs onto the agar, thePhe reverse the inhibition of bacterial growth thereby yielding an areaof bacterial growth that can be correlated to phenylalanineconcentration by comparison to similar assays performed using discscontaining known amounts of Phe.

Other methods of quantifying Phe concentration include HPLC, massspectrometry, thin layer chromatography and the like. Such methods canbe used to determine the plasma Phe concentration of a patient beforethe therapy and to monitor the Phe concentration during the therapeuticregimen to determine the efficacy thereof.

It is contemplated that the plasma Phe levels of the patients will bemonitored at convenient intervals (e.g., daily, every other day orweekly) throughout the time course of the therapeutic regimen. Bymonitoring the plasma Phe levels with such regularity, the clinicianwill be able to assess the efficacy of the treatment and adjust the BH4and/or dietary protein requirements accordingly.

IV. COMBINATION THERAPY

Certain methods of the invention involve the combined use of BH4 anddietary protein restriction to effect a therapeutic outcome in patientswith various forms of HPA. To achieve the appropriate therapeuticoutcome in the combination therapies contemplated herein, one wouldgenerally administer to the subject the BH4 composition and the dietaryrestriction in a combined amount effective to produce the desiredtherapeutic outcome (i.e., a lowering of plasma Phe concentration and/orthe ability to tolerate greater amounts of Phe/protein intake withoutproducing a concomitant increase in plasma Phe concentrations). Thisprocess may involve administering the BH4 composition and the dietaryprotein therapeutic composition at the same time. This may be achievedby administering a single composition or pharmacological proteinformulation that includes all of the dietary protein requirements andalso includes the BH4 within said protein formulation. Alternatively,the dietary protein (supplement or normal protein meal) is taken atabout the same time as a pharmacological formulation (tablet, injectionor drink) of BH4. The BH4 also may be formulated into a protein bar orother foodstuff such as brownies, pancakes, cake, suitable foringestion.

In other alternatives, the BH4 treatment may precede or follow thedietary protein therapy by intervals ranging from minutes to hours. Inembodiments where the protein and the BH4 compositions are administeredseparately, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the BH4 willstill be able to exert an advantageously effect on the patient. In suchinstances, it is contemplated that one would administer the BH4 withinabout 2-6 hours (before or after) of the dietary protein intake, with adelay time of only about 1 hour being most preferred. In certainembodiments, it is contemplated that the BH4 therapy will be acontinuous therapy where a daily dose of BH4 is administered to thepatient indefinitely. In other situations, e.g., in pregnant womenhaving only the milder forms of PKU and HPA, it may be that the BH4therapy is only continued for as long as the woman is pregnant and/orbreast feeding.

Further, in addition to therapies based solely on the delivery of BH4and dietary protein regulation, the methods of the present inventionalso contemplate combination therapy with a third composition thatspecifically targets one or more of the symptoms of HPA. For example, itis known that the deficit in tyrosine caused by HPA results in adeficiency in neurotransmitters dopamine and serotonin. Thus, in thecontext of the present invention, it is contemplated that BH4 anddietary protein based methods could be further combined withadministration of L-dopa, carbidopa and 5-hydroxytryptophanneurotransmitters to correct the defects that result from decreasedamounts of tyrosine in the diet.

In addition, gene therapy with both PAH (Christensen et al., Mol. Gent.And Metabol. 76: 313-318, 2002; Christensen et al., Gene Therapy,7:1971-1978, 2000) and phenylalanine ammonia-lyase (PAL Liu et al.,Arts. Cells. Blood. Subs and Immob. Biotech. 30(4)243-257, 2002) hasbeen contemplated by those of skill in the art. Such gene therapytechniques could be used in combination with the combined BH4/dietaryprotein restriction based therapies of the invention. In furthercombination therapies, it is contemplated that phenylase may be providedas an injectable enzyme to destroy lower Phe concentrations in thepatient. As the administration of phenylase would not generate tyrosine(unlike administration of PAH), such treatment will still result intyrosine being an essential amino acid for such patients. Thereforedietary supplementation with tyrosine may be desirable for patientsreceiving phenylase in combination with the BH4 therapy.

V. PHARMACEUTICAL COMPOSITIONS

Pharmaceutical compositions for administration according to the presentinvention can comprise a first composition comprising BH4 in apharmaceutically acceptable form optionally combined with apharmaceutically acceptable carrier. These compositions can beadministered by any means that achieve their intended purposes. Amountsand regimens for the administration of a composition according to thepresent invention can be determined readily by those with ordinary skillin the art for treating PKU. As discussed above, those of skill in theart could initially employ amounts and regimens of BH4 currently beingproposed in a medical context, e.g., those compositions that are beingproposed for modulating NOS activity, or for use in the treatment ofpain or depression as discussed in the patents listed in Section IIabove. Any of the protocols, formulations, routes of administration andthe like described that have been used for administering BH4 for loadingtests can readily be modified for use in the present invention.

The compositions and methods described herein are not limited to the useof a particular form of BH4, or form of an analog or derivative of BH4.Indeed, it is contemplated that the compositions and methods within thescope of this invention include all compositions comprising any formBH4, and any form of an analog or derivative thereof in an amounteffective to achieve its intended purpose. Nonlimiting examples ofanalogs for use in the compositions and methods described herein includepteridine, pterin, neopterin, biopterin, 7,8-Dihydrobiopterin,6-methyltetrahydropterin, and other 6-substituted tetrahydropterin andother 6-substituted tetrahydropterins, sepiapterin,6,7-Dimethyltetrahydropterin, 6-methyl biopterin and other 6-substitutedbiopterins, and other analogs that are described in the art. Nonlimitingexamples of derivatives for use in the compositions and methodsdescribed herein include the derivatives described in U.S. Pat. Nos.4,758,571; 4,774,244; 6,162,806; 5,902, 810; 2,955,110; 2,541,717;2,603,643; and 4,371,514, the disclosures of which are herebyincorporated herein.

Certain therapeutic methods of the present invention contemplate acombination therapy in which BH4-based compositions are administered inaddition to a modified protein diet, the pharmaceutical compositions ofthe invention also contemplate all compositions comprising at leastBH4-based therapeutic agent, analog or homologue thereof in an amounteffective to achieve the amelioration of one or more of the symptoms ofPKU when administered in combination with the modified protein diet. Ofcourse, the most obvious symptom that may be alleviated is that thecombined therapy produces a decrease in the plasma Phe concentration,however, other symptoms such as changes in IQ, executive function,concentration, mood, behavioral stability job performance and the likealso may be monitored. Such indicia are monitored using techniques knownto those of skill in the art.

Crystalh Polymorphs of (6R) L-Tetrahydrobiopterin Dihydrochloride Salt

It has been found that BH4, and in particular, the dihydrochloride saltof BH4, exhibits crystal polymorphism. The structure of BH4 is shownbelow:

The (6R) form of BH4 is the known biologically active form, however, BH4is also known to be unstable at ambient temperatures. It has been foundthat one crystal polymorph of BH4 is more stable, and is stable todecomposition under ambient conditions.

BH4 is difficult to handle and it is therefore produced and offered asits dihydrochloride salt (Schircks Laboratories, Jona, Switzerland) inampoules sealed under nitrogen to prevent degradation of the substancedue to its hygroscopic nature and sensitivity to oxidation. U.S. Pat.No. 4,649,197 discloses that separation of (6R)- and6(S)-L-erythro-tetrahydrobiopterin dihydrochloride into itsdiastereomers is difficult due to the poor crystallinity of6(R,S)-L-erythro-tetrahydrobiopterin dihydrochloride. The Europeanpatent number 0 079 574 describes the preparation oftetrahydrobiopterin, wherein a solid tetrahydrobiopterin dihydrochlorideis obtained as an intermediate. S. Matsuura et al. describes inChemistry Letters 1984, pages 735-738 and Heterocycles, Vol. 23, No. 12,1985 pages 3115-3120 6(R)-tetrahydrobiopterin dihydrochloride as acrystalline solid in form of colorless needles, which are characterizedby X-ray analysis disclosed in J. Biochem. 98, 1341-1348 (1985). Anoptical rotation of 6.81° was found the crystalline product, which isquite similar to the optical rotation of 6.51° reported for acrystalline solid in form of white crystals in example 6 of EP-A2-0 191335.

Results obtained during development of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride indicated that thecompound may exist in different crystalline forms, including polymorphicforms and solvates. The continued interest in this area requires anefficient and reliable method for the preparation of the individualcrystal forms of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride andcontrolled crystallization conditions to provide crystal forms, that arepreferably stable and easy to handle and to process in the manufactureand preparation of formulations, and that provide a high storagestability in substance form or as formulated product, or which provideless stable forms suitable as intermediates for controlledcrystallization for the manufacture of stable forms.

Polymorph Form B

The crystal polymorph that has been found to be the most stable isreferred to herein as “form B,” or alternatively as “polymorph B.”Results obtained during investigation and development of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride development revealedthat there are several known crystalline solids have been prepared, butnone have recognized the polymorphism and its effect on the stability ofthe BH4 crystals.

Polymorph B is a slightly hygroscopic anhydrate with the highestthermodynamic stability above about 20° C. Furthermore, form B can beeasily processed and handled due to its thermal stability, possibilityfor preparation by targeted conditions, its suitable morphology andparticle size. Melting point is near 260° C. (ΔHf>140 J/g), but no clearmelting point can be detected due to decomposition prior and duringmelting. These outstanding properties renders polymorph form Bespecially feasible for pharmaceutical application, which are preparedat elevated temperatures. Polymorph B can be obtained as a fine powderwith a particle size that may range from 0.2 μm to 500 μm.

Form B exhibits an X-ray powder diffraction pattern, expressed ind-values (A) at: 8.7 (vs), 6.9 (w), 5.90 (vw), 5.63 (m), 5.07 (m), 4.76(m), 4.40 (m), 4.15 (w), 4.00 (s), 3.95 (m), 3.52 (m), 3.44 (w), 3.32(m), 3.23 (s), 3.17 (w), 3.11 (vs), 3.06 (w), 2.99 (w), 2.96 (w), 2.94(m), 2.87 (w), 2.84 (s), 2.82 (m), 2.69 (w), 2.59 (w), 2.44 (w). FIG. 1is a graph of the characteristic X-ray diffraction pattern exhibited byform B of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

As used herein, the following the abbreviations in brackets mean:(vs)=very strong intensity; (s)=strong intensity; (m)=medium intensity;(w)=weak intensity; and (vw)=very weak intensity. A characteristic X-raypowder diffraction pattern is exhibited in FIG. 1.

It has been found that other polymorphs of BH4 have a satisfactorychemical and physical stability for a safe handling during manufactureand formulation as well as providing a high storage stability in itspure form or in formulations. In addition, it has been found that formB, and other polymorphs of BH4 can be prepared in very large quantities(e.g., 100 kilo scale) and stored over an extended period of time.

All crystal forms (polymorphs, hydrates and solvates), inclusive crystalform B, can be used for the preparation of the most stable polymorph B.Polymorph B may be obtained by phase equilibration of suspensions ofamorphous or other forms than polymorph form B, such as polymorph A, insuitable polar and non aqueous solvents. Thus, the pharmaceuticalpreparations described herein refers to a preparation of polymorph formB of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Other forms of BH4 can be converted for form B by dispersing the otherform of BH4 in a solvent at room temperature, stirring the suspension atambient temperatures for a time sufficient to produce polymorph form B,thereafter isolating crystalline form B and removing the solvent fromthe isolated form B. Ambient temperatures, as used herein, meantemperatures in a range from 0° C. to 60° C., preferably 15° C. to 40°C. The applied temperature may be changed during treatment and stirringby decreasing the temperature stepwise or continuously. Suitablesolvents for the conversion of other forms to form B include but are notlimited to, methanol, ethanol, isopropanol, other C3- and C4-alcohols,acetic acid, acetonitrile, tetrahydrofurane, methy-t-butyl ether,1,4-dioxane, ethyl acetate, isopropyl acetate, other C3-C6-acetates,methyl ethyl ketone and other methyl-C3-C5 alkyl-ketones. The time tocomplete phase equilibration may be up to 30 hours and preferably up to20 hours or less than 20 hours.

Polymorph B may also be obtained by crystallisation from solventmixtures containing up to about 5% water, especially from mixtures ofethanol, acetic acid and water. It has been found that polymorph form Bof (6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bydissolution, optionally at elevated temperatures, preferably of a solidlower energy form than form B or of form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a solvent mixturecomprising ethanol, acetic acid and water, addition of seeds to thesolution, cooling the obtained suspension and isolation of the formedcrystals. Dissolution may be carried out at room temperature or up to70° C., preferably up to 50° C. There may be used the final solventmixture for dissolution or the starting material may be first dissolvedin water and the other solvents may than be added both or one after theother solvent. The composition of the solvent mixture may comprise avolume ratio of water:acetic acid:tetrahydrofuran of 1:3:2 to 1:9:4 andpreferably 1:5:4. The solution is preferably stirred. Cooling may meantemperatures down to −40° C. to 0° C., preferably down to 10° C. to 30°C. Suitable seeds are polymorph form B from another batch or crystalshaving a similar or identical morphology. After isolation, thecrystalline form B can be washed with a non-solvent such as acetone ortetrahydrofurane and dried in usual manner.

Polymorph B may also be obtained by crystallisation from aqueoussolutions through the addition of non-solvents such as methanol, ethanoland acetic acid. The crystallisation and isolation procedure can beadvantageously carried out at room temperature without cooling thesolution. This process is therefore very suitable to be carried out atan industrial scale.

In one embodiment of the compositions and methods described herein, acomposition including polymorph form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is prepared bydissolution of a solid form other than form B or of form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in water at ambienttemperatures, adding a non-solvent in an amount sufficient to form asuspension, optionally stirring the suspension for a certain time, andthereafter isolation of the formed crystals. The composition is furthermodified into a pharmaceutical composition as described below.

The concentration of (6R)-L-erythro-tetrahydrobiopterin dihydrochloridein the aqueous solution may be from 10 to 80 percent by weight, morepreferably from 20 to 60 percent by weight, by reference to thesolution. Preferred non-solvents (i.e., solvents useful in preparingsuspensions of BH4) are methanol, ethanol and acetic acid. Thenon-solvent may be added to the aqueous solution. More preferably, theaqueous solution is added to the non-solvent. The stirring time afterformation of the suspension may be up to 30 hours and preferably up to20 hours or less than 20 hours. Isolation by filtration and drying iscarried out in known manner as described above.

Polymorph form B is a very stable crystalline form, that can be easilyfiltered off, dried and ground to particle sizes desired forpharmaceutical formulations. These outstanding properties renderspolymorph form B especially feasible for pharmaceutical application.

Polymorph Form A

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form A,” or “polymorph A.”Polymorph A is slightly hygroscopic and adsorbs water to a content ofabout 3 percent by weight, which is continuously released between 50° C.and 200° C., when heated at a rate of 10° C./minute. The polymorph A isa hygroscopic anhydrate which is a meta-stable form with respect to formB; however, it is stable over several months at ambient conditions ifkept in a tightly sealed container. Form A is especially suitable asintermediate and starting material to produce stable polymorph forms.Polymorph form A can be prepared as a solid powder with desired mediumparticle size range which is typically ranging from 1 μm to about 500μm.

Polymorph A which exhibits a characteristic X-ray powder diffractionpattern with characteristic peaks expressed in d-values (A) of: 15.5(vs), 12.0 (m), 6.7 (m), 6.5 (m), 6.3 (w), 6.1 (w), 5.96 (w), 5.49 (m),4.89 (m), 3.79 (m), 3.70 (s), 3.48 (m), 3.45 (m), 3.33 (s), 3.26 (s),3.22 (m), 3.18 (m), 3.08 (m), 3.02 (w), 2.95 (w), 2.87 (m), 2.79 (w),2.70 (w). FIG. 2 is a graph of the characteristic X-ray diffractionpattern exhibited by form A of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

Polymorph A exhibits a characteristic Raman spectra bands, expressed inwave numbers (cm-1) at: 2934 (w), 2880 (w), 1692 (s), 1683 (m), 1577(w), 1462 (m), 1360 (w), 1237 (w), 1108 (w), 1005 (vw), 881 (vw), 813(vw), 717 (m), 687 (m), 673 (m), 659 (m), 550 (w), 530 (w), 492 (m), 371(m), 258 (w), 207 (w), 101 (s), 87 (s) cm-1.

Polymorph form A may be obtained by freeze drying or water removal ofsolutions of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride inwater. Polymorph form A of (6R)-L-erythro-tetrahydrobiopterindihydrochloride can be prepared by dissolving(6R)-L-erythro-tetrahydrobiopterin dihydrochloride at ambienttemperatures in water, (1) cooling the solution to low temperatures forsolidifying the solution, and removing water under reduced pressure, or(2) removing water from said aqueous solution.

The crystalline form A can be isolated by filtration and then dried toevaporate absorbed water from the product. Drying conditions and methodsare known and drying of the isolated product or water removal pursuantto variant (2) described herein may be carried out in applying elevatedtemperatures, for example up to 80° C., preferably in the range from 30°C. to 80° C., under vacuum or elevated temperatures and vacuum. Prior toisolation of a precipitate obtained in variant (2), the suspension maybe stirred for a certain time for phase equilibration. The concentrationof (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in the aqueoussolution may be from 5 to 40 percent by weight, by reference to thesolution.

A fast cooling is preferred to obtain solid solutions as startingmaterial. A reduced pressure is applied until the solvent is completelyremoved. Freeze drying is a technology well known in the art. The timeto complete solvent removal is dependent on the applied vacuum, whichmay be from 0.01 to 1 mbar, the solvent used and the freezingtemperature.

Polymorph form A is stable at room temperature or below room temperatureunder substantially water free conditions, which is demonstrated withphase equilibration tests of suspensions in tetrahydrofuran ortertiary-butyl methyl ether stirred for five days and 18 hoursrespectively under nitrogen at room temperature. Filtration and airdrying at room temperature yields unchanged polymorph form A.

Polymorph Form F

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form F,” or “polymorph F.”Polymorph F is slightly hygroscopic and adsorbs water to a content ofabout 3 percent by weight, which is continuously released between 50° C.and 200° C., when heated at a rate of 10° C./minute. The polymorph F isa meta-stable form and a hygroscopic anhydrate, which is more stablethan form A at ambient lower temperatures and less stable than form B athigher temperatures and form F is especially suitable as intermediateand starting material to produce stable polymorph forms. Polymorph formF can be prepared as a solid powder with desired medium particle sizerange which is typically ranging from 1 μm to about 500 μm.

Polymorph F exhibits a characteristic X-ray powder diffraction patternwith characteristic peaks expressed in d-values (Å) at: 17.1 (vs), 12.1(w), 8.6 (w), 7.0 (w), 6.5 (w), 6.4 (w), 5.92 (w), 5.72 (w), 5.11 (w),4.92 (m), 4.86 (w), 4.68 (m), 4.41 (w), 4.12 (w), 3.88 (w), 3.83 (w),3.70 (m), 3.64 (w), 3.55 (m), 3.49 (s), 3.46 (vs), 3.39 (s), 3.33 (m),3.31 (m), 3.27 (m), 3.21 (m), 3.19 (m), 3.09 (m), 3.02 (m), and 2.96(m). FIG. 3 is a graph of the characteristic X-ray diffraction patternexhibited by form F of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

Polymorph F may be obtained by phase equilibration of suspensions ofpolymorph form A in suitable polar and non-aqueous solvents, whichscarcely dissolve said lower energy forms, especially alcohols such asmethanol, ethanol, propanol and isopropanol. Polymorph form F of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can also be preparedby dispersing particles of solid form A of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a non-aqueoussolvent that scarcely dissolves said (6R)-L-erythro-tetrahydrobiopterindihydrochloride below room temperature, stirring the suspension at saidtemperatures for a time sufficient to produce polymorph form F,thereafter isolating crystalline form F and removing the solvent fromthe isolated form F. Removing of solvent and drying may be carried outunder air, dry air or a dry protection gas such as nitrogen or noblegases and at or below room temperature, for example down to 0° C. Thetemperature during phase equilibration is preferably from 5 to 15° C.and most preferably about 10° C.

Polymorph Form J

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form J,” or “polymorph J.” Thepolymorph J is slightly hygroscopic and adsorbs water when handled atair humidity. The polymorph J is a meta-stable form and a hygroscopicanhydrate, and it can be transformed back into form E described below,from which it is obtained upon exposure to high relative humidityconditions such as above 75% relative humidity. Form J is especiallysuitable as intermediate and starting material to produce stablepolymorph forms. Polymorph form J can be prepared as a solid powder withdesired medium particle size range which is typically ranging from 1 μmto about 500 μm.

Form J exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.6 (m), 6.6 (w),6.4 (w), 5.47 (w), 4.84 (w), 3.29 (vs), and 3.21 (vs). FIG. 4 is a graphof the characteristic X-ray diffraction pattern exhibited by form J of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Polymorph J may be obtained by dehydration of form E at moderatetemperatures under vacuum. In particular, polymorph form J of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bytaking form E and removing the water from form E by treating form E in avacuum drier to obtain form J at moderate temperatures which may mean atemperature in the range of 25 to 70° C., and most preferably 30 to 50°C.

Polymorph Form K

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form K,” or “polymorph K.”Polymorph K is slightly hygroscopic and adsorbs water to a content ofabout 2.0 percent by weight, which is continuously released between 50°C. and 100° C., when heated at a rate of 10° C./minute. The polymorph Kis a meta-stable form and a hygroscopic anhydrate, which is less stablethan form B at higher temperatures and form K is especially suitable asintermediate and starting material to produce stable polymorph forms, inparticular form B. Polymorph form K can be prepared as a solid powderwith desired medium particle size range which is typically ranging from1 μm to about 500 μm.

Form K exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.0 (s), 9.4 (w),6.6 (w), 6.4 (w), 6.3 (w), 6.1 (w), 6.0 (w), 5.66 (w), 5.33 (w), 5.13(vw), 4.73 (m), 4.64 (m), 4.48 (w), 4.32 (vw), 4.22 (w), 4.08 (w), 3.88(w), 3.79 (w), 3.54 (m), 3.49 (vs), 3.39 (m), 3.33 (vs), 3.13 (s), 3.10(m), 3.05 (m), 3.01 (m), 2.99 (m), and 2.90 (m). FIG. 5 is a graph ofthe characteristic X-ray diffraction pattern exhibited by form K of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Polymorph K may be obtained by crystallization from mixtures of polarsolvents containing small amounts of water and in the presence of smallamounts of ascorbic acid. Solvents for the solvent mixture may beselected from acetic acid and an alcohol such as methanol, ethanol, n-or isopropanol. In particular, polymorph form K of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bydissolving (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in amixture of acetic acid and an alcohol or tetrahydrofuran containingsmall amounts of water and a small amount of ascorbic acid at elevatedtemperatures, lowering temperature below room temperature to crystallizesaid dihydrochloride, isolating the precipitate and drying the isolatedprecipitate at elevated temperature optionally under vacuum. Suitablealcohols are for example methanol, ethanol, propanol and isopropanol,whereby ethanol is preferred. The ratio of acetic acid to alcohol ortetrahydrofuran may be from 2:1 to 1:2 and preferably about 1:1.Dissolution of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride can becarried out in presence of a higher water content and more of theanti-solvent mixture can be added to obtain complete precipitation. Theamount of water in the final composition may be from 0.5 to 5 percent byweight and the amount of ascorbic acid may be from 0.01 to 0.5 percentby weight, both by reference to the solvent mixture. The temperature fordissolution may be in the range from 30 to 100 and preferably 35 to 70°C. and the drying temperature may be in the range from 30 to 50° C. Theprecipitate may be washed with an alcohol such as ethanol afterisolation, e.g., filtration. The polymorph K can easily be converted inthe most stable form B by phase equilibration in e.g., isopropanol andoptionally seeding with form B crystals at above room temperature suchas temperatures from 30 to 40° C.

Hydrate Forms of (6R) L-Tetrahydrobiopterin Dihydrochloride Salt

As further described below, it has been found that(6R)-L-erythro-tetrahydrobiopterin dihydrochloride exists as a number ofcrystalline hydrate, which shall be described and defined herein asforms C, D, E, H, and O. These hydrate forms are useful as a stable formof BH4 for the pharmaceutical preparations described herein and in thepreparation of compositions including stable crystal polymorphs of BH4.

Hydrate Form C

It has been found that a hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form C,” or “hydrate C.” Thehydrate form C is slightly hygroscopic and has a water content ofapproximately 5.5 percent by weight, which indicates that form C is amonohydrate. The hydrate C has a melting point near 94° C. (ΔH_(f) isabout 31 J/g) and hydrate form C is especially suitable as intermediateand starting material to produce stable polymorphic forms. Polymorphform C can be prepared as a solid powder with desired medium particlesize range which is typically ranging from 1 μm to about 500 μm.

Form C exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 18.2 (m), 15.4 (w),13.9 (vs), 10.4 (w), 9.6 (w), 9.1 (w), 8.8 (m), 8.2 (w), 8.0 (w), 6.8(m), 6.5 (w), 6.05 (m), 5.77 (w), 5.64 (w), 5.44 (w), 5.19 (w), 4.89(w), 4.76 (w), 4.70 (w), 4.41 (w), 4.25 (m), 4.00 (m), 3.88 (m), 3.80(m), 3.59 (s), 3.50 (m), 3.44 (m), 3.37 (m), 3.26 (s), 3.19 (vs), 3.17(s), 3.11 (m), 3.06 (m), 3.02 (m), 2.97 (vs), 2.93 (m), 2.89 (m), 2.83(m), and 2.43 (m). FIG. 6 is a graph of the characteristic X-raydiffraction pattern exhibited by hydrate form C of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form C may be obtained by phase equilibration at ambienttemperatures of a polymorph form such as polymorph B suspension in anon-solvent which contains water in an amount of preferably about 5percent by weight, by reference to the solvent. Hydrate form C of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride cab be prepared bysuspending (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in anon-solvent such as, heptane, C1-C4-alcohols such as methanol, ethanol,1- or 2-propanol, acetates, such as ethyl acetate, acetonitrile, aceticacid or ethers such as terahydrofuran, dioxane, tertiary-butyl methylether, or binary or ternary mixtures of such non-solvents, to whichsufficient water is added to form a monohydrate, and stirring thesuspension at or below ambient temperatures (e.g., 0 to 30° C.) for atime sufficient to form a monohydrate. Sufficient water may mean from 1to 10 and preferably from 3 to 8 percent by weight of water, byreference to the amount of solvent. The solids may be filtered off anddried in air at about room temperature. The solid can absorb some waterand therefore possess a higher water content than the theoretical valueof 5.5 percent by weight. Hydrate form C is unstable with respect toforms D and B, and easily converted to polymorph form B at temperaturesof about 40° C. in air and lower relative humidity. Form C can betransformed into the more stable hydrate D by suspension equilibrationat room temperature.

Hydrate Form D

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form D,” or “hydrate D.” Thehydrate form D is slightly hygroscopic and may have a water content ofapproximately 5.0 to 7.0 percent by weight, which suggests that form Dis a monohydrate. The hydrate D has a melting point near 153° C. (ΔH_(f)is about 111 J/g) and is of much higher stability than form C and iseven stable when exposed to air humidity at ambient temperature. Hydrateform D can therefore either be used to prepare formulations or asintermediate and starting material to produce stable polymorph forms.Polymorph form D can be prepared as a solid powder with desired mediumparticle size range which is typically ranging from 1 μm to about 500

Form D exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 8.6 (s), 6.8 (w),5.56 (m), 4.99 (m), 4.67 (s), 4.32 (m), 3.93 (vs), 3.88 (w), 3.64 (w),3.41 (w), 3.25 (w), 3.17 (m), 3.05 (s), 2.94 (w), 2.92 (w), 2.88 (m),2.85 (w), 2.80 (w), 2.79 (m), 2.68 (w), 2.65 (w), 2.52 (vw), 2.35 (w),2.34 (w), 2.30 (w), and 2.29 (w). FIG. 7 is a graph of thecharacteristic X-ray diffraction pattern exhibited by hydrate form D of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form D may be obtained by adding at about room temperatureconcentrated aqueous solutions of (6R)-L-erythro-tetrahydrobiopterindihydrochloride to an excess of a non-solvent such as hexane, heptane,dichloromethane, 1- or 2-propanol, acetone, ethyl acetate, acetonitrile,acetic acid or ethers such as terahydrofuran, dioxane, tertiary-butylmethyl ether, or mixtures of such non-solvents, and stirring thesuspension at ambient temperatures. The crystalline solid can befiltered off and then dried under dry nitrogen at ambient temperatures.A preferred non-solvent is isopropanol. The addition of the aqueoussolution may carried out drop-wise to avoid a sudden precipitation.Hydrate form D of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride canbe prepared by adding at about room temperature a concentrated aqueoussolutions of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride to anexcess of a non-solvent and stirring the suspension at ambienttemperatures. Excess of non-solvent may mean a ratio of aqueous to thenon solvent from 1:10 to 1:1000. Form D contains a small excess ofwater, related to the monohydrate, and it is believed that it isabsorbed water due to the slightly hygroscopic nature of thiscrystalline hydrate. Hydrate form D is deemed to be the most stable oneunder the known hydrates at ambient temperatures and a relative humidityof less than 70%. Hydrate form D may be used for formulations preparedunder conditions, where this hydrate is stable. Ambient temperature maymean 20 to 30° C.

Hydrate Form E

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form E,” or “hydrate E.” Thehydrate form E has a water content of approximately 10 to 14 percent byweight, which suggests that form E is a dihydrate. The hydrate E isformed at temperatures below room temperature. Hydrate form E isespecially suitable as intermediate and starting material to producestable polymorph forms. It is especially suitable to produce thewater-free form J upon drying under nitrogen or optionally under vacuum.Form E is non-hygroscopic and stable under rather high relativehumidities, i.e., at relative humidities above about 60% and up to about85%. Polymorph form E can be prepared as a solid powder with desiredmedium particle size range which is typically ranging from 1 μm to about500 μm.

Form E exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 15.4 (s), 6.6 (w),6.5 (w), 5.95 (vw), 5.61 (vw), 5.48 (w), 5.24 (w), 4.87 (w), 4.50 (vw),4.27 (w), 3.94 (w), 3.78 (w), 3.69 (m), 3.60 (w), 3.33 (s), 3.26 (vs),3.16 (w), 3.08 (m), 2.98 (w), 2.95 (m), 2.91 (w), 2.87 (m), 2.79 (w),2.74 (w), 2.69 (w), and 2.62 (w). FIG. 8 is a graph of thecharacteristic X-ray diffraction pattern exhibited by hydrate form E of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form E may be obtained by adding concentrated aqueous solutionsof (6R)-L-erythro-tetrahydrobiopterin dihydrochloride to an excess of anon-solvent cooled to temperatures from about 10 to −10° C. andpreferably between 0 to 10° C. and stirring the suspension at saidtemperatures. The crystalline solid can be filtered off and then driedunder dry nitrogen at ambient temperatures. Non-solvents are for examplesuch as hexane, heptane, dichloromethane, 1- or 2-propanol, acetone,ethyl acetate, acetonitrile, acetic acid or ethers such asterahydrofuran, dioxane, tertiary-butyl methyl ether, or mixtures ofsuch non-solvents. A preferred non-solvent is isopropanol. The additionof the aqueous solution may carried out drop-wise to avoid a suddenprecipitation. Hydrate form E of (6R)-L-erythro-tetrahydrobiopterindihydrochloride can be prepared by adding a concentrated aqueoussolutions of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride to anexcess of a non-solvent which is cooled to temperatures from about 10 to−10° C., and stirring the suspension at ambient temperatures. Excess ofnon-solvent may mean a ratio of aqueous to the non solvent from 1:10 to1:1000. A preferred non-solvent is tetrahydrofuran. Another preparationprocess comprises exposing polymorph form B to an air atmosphere with arelative humidity of 70 to 90%, preferably about 80%. Hydrate form E isdeemed to be a dihydrate, whereby some additional water may be absorbed.Polymorph form E can be transformed into polymorph J upon drying undervacuum at moderate temperatures, which may mean between 20° C. and 50°C. at pressures between 0 and 100 mbar. Form E is especially suitablefor formulations in semi solid forms because of its stability at highrelative humidities.

Hydrate Form H

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form H,” or “hydrate H.” Thehydrate form H has a water content of approximately 5.0 to 7.0 percentby weight, which suggests that form H is a hygroscopic monohydrate. Thehydrate form H is formed at temperatures below room temperature. Hydrateform H is especially suitable as intermediate and starting material toproduce stable polymorph forms. Polymorph form H can be prepared as asolid powder with desired medium particle size range which is typicallyranging from 1 μm to about 500

Form H exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 8.6 15.8 (vs), 10.3(w), 8.0 (w), 6.6 (w), 6.07 (w), 4.81 (w), 4.30 (w), 3.87 (m), 3.60 (m),3.27 (m), 3.21 (m), 3.13 (w), 3.05 (w), 2.96 (m), 2.89 (m), 2.82 (w),and 2.67 (m). FIG. 9 is a graph of the characteristic X-ray diffractionpattern exhibited by hydrate form H of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form H may be obtained by dissolving at ambient temperatures(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a mixture ofacetic acid and water, adding then a non-solvent to precipitate acrystalline solid, cooling the obtained suspension and stirring thecooled suspension for a certain time. The crystalline solid is filteredoff and then dried under vacuum at ambient temperatures. Non-solventsare for example such as hexane, heptane, dichloromethane, 1- or2-propanol, acetone, ethyl acetate, acetonitril, acetic acid or etherssuch as terahydrofuran, dioxane, tertiary-butyl methyl ether, ormixtures of such non-solvents. A preferred non-solvent istetrahydrofuran. Hydrate form H of (6R)-L-erythro-tetrahydrobiopterindihydrochloride can be by prepared by dissolving at ambient temperatures(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a mixture ofacetic acid and a less amount than that of acetic acid of water, addinga non-solvent and cooling the obtained suspension to temperatures in therange of −10 to 10° C., and preferably −5 to 5° C., and stirring thesuspension at said temperature for a certain time. Certain time may mean1 to 20 hours. The weight ratio of acetic acid to water may be from 2:1to 25:1 and preferably 5:1 to 15:1. The weight ratio of aceticacid/water to the non-solvent may be from 1:2 to 1:5. Hydrate form Hseems to be a monohydrate with a slight excess of water absorbed due tothe hygroscopic nature.

Hydrate Form O

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form O,” or “hydrate O.” Thehydrate form O is formed at temperatures near room temperature. Hydrateform O is especially suitable as intermediate and starting material toproduce stable polymorph forms. Polymorph form O can be prepared as asolid powder with desired medium particle size range which is typicallyranging from 1 μm to about 500 μm.

Form O exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 15.9 (w), 14.0 (w),12.0 (w), 8.8 (m), 7.0 (w), 6.5 (w), 6.3 (m), 6.00 (w), 5.75 (w), 5.65(m), 5.06 (m), 4.98 (m), 4.92 (m), 4.84 (w), 4.77 (w), 4.42 (w), 4.33(w), 4.00 (m), 3.88 (m), 3.78 (w), 3.69 (s), 3.64 (s), 3.52 (vs), 3.49(s), 3.46 (s), 3.42 (s), 3.32 (m), 3.27 (m), 3.23 (s), 3.18 (s), 3.15(vs), 3.12 (m), 3.04 (vs), 2.95 (m), 2.81 (s), 2.72 (m), 2.67 (m), and2.61 (m). FIG. 10 is a graph of the characteristic X-ray diffractionpattern exhibited by hydrate form O of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form O can be prepared by exposure of polymorphic form F to anitrogen atmosphere containing water vapor with a resulting relativehumidity of about 52% for about 24 hours. The fact that form F, which isa slightly hygroscopic anhydrate, can be used to prepare form O under52% relative humidity suggests that form O is a hydrate, which is morestable than form F under ambient temperature and humidity conditions.

Solvate Forms of (6R) L-Tetrahydrobiopterin Dihydrochloride Salt

As further described below, it has been found that(6R)-L-erythro-tetrahydrobiopterin dihydrochloride exists as a number ofcrystalline solvate forms, which shall be described and defined hereinas forms G, I, L, M, and N. These solvate forms are useful as a stableform of BH4 for the pharmaceutical preparations described herein and inthe preparation of compositions including stable crystal polymorphs ofBH4.

Solvate Form G

It has been found that an ethanol solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form G,” or “hydrate G.” Theethanol solvate form G has a ethanol content of approximately 8.0 to12.5 percent by weight, which suggests that form G is a hygroscopic monoethanol solvate. The solvate form G is formed at temperatures below roomtemperature. Form G is especially suitable as intermediate and startingmaterial to produce stable polymorph forms. Polymorph form G can beprepared as a solid powder with a desired medium particle size rangewhich is typically ranging from 1 μm to about 500 μm.

Form G exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.5 (vs), 10.9 (w),9.8 (w), 7.0 (w), 6.3 (w), 5.74 (w), 5.24 (vw), 5.04 (vw), 4.79 (w),4.41 (w), 4.02 (w), 3.86 (w), 3.77 (w), 3.69 (w), 3.63 (m), 3.57 (m),3.49 (m), 3.41 (m), 3.26 (m), 3.17 (m), 3.07 (m), 2.97 (m), 2.95 (m),2.87 (w), and 2.61 (w). FIG. 11 is a graph of the characteristic X-raydiffraction pattern exhibited by solvate form G of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Ethanol solvate form G may be obtained by crystallization ofL-erythro-tetrahydrobiopterin dihydrochloride dissolved in water andadding a large excess of ethanol, stirring the obtained suspension at orbelow ambient temperatures and drying the isolated solid under air ornitrogen at about room temperature. Here, a large excess of ethanolmeans a resulting mixture of ethanol and water with less than 10% water,preferably about 3 to 6%. Ethanolate form G of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bydissolving at about room temperature to temperatures of 75° C.(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in water or in amixture of water and ethanol, cooling a heated solution to roomtemperature and down to 5 to 10° C., adding optionally ethanol tocomplete precipitation, stirring the obtained suspension at temperaturesof 20 to 5° C., filtering off the white, crystalline solid and dryingthe solid under air or a protection gas such as nitrogen at temperaturesabout room temperature. The process may be carried out in a firstvariant in dissolving (6R)-L-erythro-tetrahydrobiopterin dihydrochlorideat about room temperature in a lower amount of water and then adding anexcess of ethanol and then stirring the obtained suspension for a timesufficient for phase equilibration. In a second variant,(6R)-L-erythro-tetrahydrobiopterin dihydrochloride may be suspended inethanol, optionally adding a lower amount of water, and heating thesuspension and dissolute (6R)-L-erythro-tetrahydrobiopterindihydrochloride, cooling down the solution to temperatures of about 5 to15° C., adding additional ethanol to the suspension and then stirringthe obtained suspension for a time sufficient for phase equilibration.

Solvate Form I

It has been found that an acetic acid solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form I,” or “hydrate I.” Theacetic acid solvate form I has an acetic acid content of approximately12.7 percent by weight, which suggests that form I is a hygroscopicacetic acid mono solvate. The solvate form I is formed at temperaturesbelow room temperature. Acetic acid solvate form I is especiallysuitable as intermediate and starting material to produce stablepolymorph forms. Polymorph form I can be prepared as a solid powder withdesired medium particle size range which is typically ranging from 1 μmto about 500 μm.

Form I exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.5 (m), 14.0 (w),11.0 (w), 7.0 (vw), 6.9 (vw), 6.2 (vw), 5.30 (w), 4.79 (w), 4.44 (w),4.29 (w), 4.20 (vw), 4.02 (w), 3.84 (w), 3.80 (w), 3.67 (vs), 3.61 (m),3.56 (w), 3.44 (m), 3.27 (w), 3.19 (w), 3.11(s), 3.00 (m), 2.94 (w),2.87 (w), and 2.80 (w). FIG. 12 is a graph of the characteristic X-raydiffraction pattern exhibited by solvate form I of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Acetic acid solvate form I may be obtained by dissolution ofL-erythro-tetrahydrobiopterin dihydrochloride in a mixture of aceticacid and water at elevated temperature, adding further acetic acid tothe solution, cooling down to a temperature of about 10° C., thenwarming up the formed suspension to about 15° C., and then stirring theobtained suspension for a time sufficient for phase equilibration, whichmay last up to 3 days. The crystalline solid is then filtered off anddried under air or a protection gas such as nitrogen at temperaturesabout room temperature.

Solvate Form L

It has been found that a mixed ethanol solvate/hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form L,” or “hydrate L.” Form Lmay contain 4% but up to 13% ethanol and 0% to about 6% of water. Form Lmay be transformed into form G when treated in ethanol at temperaturesfrom about 0° C. to 20° C. In addition form L may be transformed intoform B when treated in an organic solvent at ambient temperatures (10°C. to 60° C.). Polymorph form L can be prepared as a solid powder withdesired medium particle size range which is typically ranging from 1 μmto about 500 μm.

Form L exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.1 (vs), 10.4 (w),9.5 (w), 9.0 (vw), 6.9 (w), 6.5 (w), 6.1 (w), 5.75 (w), 5.61 (w), 5.08(w), 4.71 (w), 3.86 (w), 3.78 (w), 3.46 (m), 3.36 (m), 3.06 (w), 2.90(w), and 2.82 (w). FIG. 13 is a graph of the characteristic X-raydiffraction pattern exhibited by solvate form L of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Form L may be obtained by suspending hydrate form E at room temperaturein ethanol and stirring the suspension at temperatures from 0 to 10° C.,preferably about 5° C., for a time sufficient for phase equilibration,which may be 10 to 20 hours. The crystalline solid is then filtered offand dried preferably under reduced pressure at 30° C. or under nitrogen.Analysis by TG-FTIR suggests that form L may contain variable amounts ofethanol and water, i.e., it can exist as an polymorph (anhydrate), as amixed ethanol solvate/hydrate, or even as a hydrate.

Solvate Form M

It has been found that an ethanol solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form M,” or “hydrate M.” Form Mmay contain 4% but up to 13% ethanol and 0% to about 6% of water, whichsuggests that form M is a slightly hygroscopic ethanol solvate. Thesolvate form M is formed at room temperature. Form M is especiallysuitable as intermediate and starting material to produce stablepolymorph forms, since form M can be transformed into form G whentreated in ethanol at temperatures between about −10° to 15° C., andinto form B when treated in organic solvents such as ethanol, C3 and C4alcohols, or cyclic ethers such as THF and dioxane. Polymorph form M canbe prepared as a solid powder with desired medium particle size rangewhich is typically ranging from 1 μm to about 500 μm.

Form M exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 18.9 (s), 6.4 (m),6.06 (w), 5.66 (w), 5.28 (w), 4.50 (w), 4.23 (w), and 3.22 (vs). FIG. 14is a graph of the characteristic X-ray diffraction pattern exhibited bysolvate form M of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Ethanol solvate form M may be obtained by dissolution ofL-erythro-tetrahydrobiopterin dihydrochloride in ethanol and evaporationof the solution under nitrogen at ambient temperature, i.e., between 10°C. and 40° C. Form M may also be obtained by drying of form G under aslight flow of dry nitrogen at a rate of about 20 to 100 ml/min.Depending on the extent of drying under nitrogen, the remaining amountof ethanol may be variable, i.e., from about 3% to 13%.

Solvate Form N

It has been found that another solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form N,” or “hydrate N.” Form Nmay contain in total up to 10% of isopropanol and water, which suggeststhat form N is a slightly hygroscopic isopropanol solvate. Form N may beobtained through washing of form D with isopropanol and subsequentdrying in vacuum at about 30° C. Form N is especially suitable asintermediate and starting material to produce stable polymorph forms.Polymorph form N can be prepared as a solid powder with desired mediumparticle size range which is typically ranging from 1 μm to about 500μm.

Form N exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 19.5 (m), 9.9 (w),6.7 (w), 5.15 (w), 4.83(w), 3.91 (w), 3.56 (m), 3.33 (vs), 3.15 (w),2.89 (w), 2.81 (w), 2.56 (w), and 2.36 (w). FIG. 15 is a graph of thecharacteristic X-ray diffraction pattern exhibited by solvate form N of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

The isopropanol form N may be obtained by dissolution ofL-erythro-tetrahydrobiopterin dihydrochloride in 4.0 ml of a mixture ofisopropanol and water (mixing volume ratio for example 4:1). To thissolution is slowly added isopropanol (IPA, for example about 4.0 ml) andthe resulting suspension is cooled to 0° C. and stirred for severalhours (e.g., about 10 to 18 hours) at this temperature. The suspensionis filtered and the solid residue washed with isopropanol at roomtemperature. The obtained crystalline material is then dried at ambienttemperature (e.g., about 20 to 30° C.) and reduced pressure (about 2 to10 mbar) for several hours (e.g., about 5 to 20 hours). TG-FTIR shows aweight loss of 9.0% between 25 to 200° C., which is attributed to bothisopropanol and water. This result suggests that form N can exist eitherin form of an isopropanol solvate, or in form of mixed isopropanolsolvate/hydrate, or as an non-solvated form containing a small amount ofwater.

For the preparation of the polymorph forms, there may be usedcrystallization techniques well known in the art, such as stirring of asuspension (phase equilibration in), precipitation, re-crystallization,evaporation, solvent like water sorption methods or decomposition ofsolvates. Diluted, saturated or super-saturated solutions may be usedfor crystallization, with or without seeding with suitable nucleatingagents. Temperatures up to 100° C. may be applied to form solutions.Cooling to initiate crystallization and precipitation down to −100° C.and preferably down to −30° C. may be applied. Meta-stable polymorphs orpseudo-polymorphic forms can be used to prepare solutions or suspensionsfor the preparation of more stable forms and to achieve higherconcentrations in the solutions.

It was surprisingly found that hydrate form D is the most stable formunder the hydrates and forms B and D are especially suitable to be usedin pharmaceutical formulations. Forms B and D presents some advantageslike an aimed manufacture, good handling due to convenient crystal sizeand morphology, very good stability under production conditions ofvarious types of formulation, storage stability, higher solubility, andhigh bio-availability. Accordingly, in a method and/or a compositiondisclosed herein the form of BH4 present in a mixture is preferably astabilized crystal form of BH4 an is selected from the group consistingof crystal polymorph form A, crystal polymorph form B, crystal polymorphform F, crystal polymorph form J, crystal polymorph form K, crystalhydrate form C, crystal hydrate form D, crystal hydrate form E, crystalhydrate form H, crystal hydrate form O, solvate crystal form G, solvatecrystal form I, solvate crystal form L, solvate crystal form M, solvatecrystal form N, and combinations thereof. More preferably, the form ofBH4 is for use in a composition and method disclosed herein ispharmaceutical composition including polymorph form B and/or hydrateform D of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride and apharmaceutically acceptable carrier or diluent.

The crystal forms of (6R)-L-erythro-tetrahydrobiopterin dihydrochloridemay be used together with folic acid or tetrahydrofolic acid or theirpharmaceutically acceptable salts such as sodium, potassium, calcium orammonium salts, each alone or additionally with arginine. The weightratio of crystal forms:folic acids or salts thereof: arginine may befrom about 1:10:10 to about 10:1:1.

VI. PHARMACEUTICAL FORMULATIONS

The formulations described herein are preferably administered as oralformulations. Oral formulations are preferably solid formulations suchas capsules, tablets, pills and troches, or liquid formulations such asaqueous suspensions, elixirs and syrups. The various form of BH4described herein can be directly used as powder (micronized particles),granules, suspensions or solutions, or it may be combined together withother pharmaceutically acceptable ingredients in admixing the componentsand optionally finely divide them, and then filling capsules, composedfor example from hard or soft gelatin, compressing tablets, pills ortroches, or suspend or dissolve them in carriers for suspensions,elixirs and syrups. Coatings may be applied after compression to formpills.

Pharmaceutically acceptable ingredients are well known for the varioustypes of formulation and may be for example binders such as natural orsynthetic polymers, excipients, lubricants, surfactants, sweetening andflavouring agents, coating materials, preservatives, dyes, thickeners,adjuvants, antimicrobial agents, antioxidants and carriers for thevarious formulation types. Nonlimiting examples of binders useful in acomposition described herein include gum tragacanth, acacia, starch,gelatine, and biological degradable polymers such as homo- orco-polyesters of dicarboxylic acids, alkylene glycols, polyalkyleneglycols and/or aliphatic hydroxyl carboxylic acids; homo- orco-polyamides of dicarboxylic acids, alkylene diamines, and/or aliphaticamino carboxylic acids; corresponding polyester-polyamide-co-polymers,polyanhydrides, polyorthoesters, polyphosphazene and polycarbonates. Thebiological degradable polymers may be linear, branched or crosslinked.Specific examples are poly-glycolic acid, poly-lactic acid, andpoly-d,l-lactide/glycolide. Other examples for polymers arewater-soluble polymers such as polyoxaalkylenes (polyoxaethylene,polyoxapropylene and mixed polymers thereof, poly-acrylamides andhydroxylalkylated polyacrylamides, poly-maleic acid and esters or-amides thereof, poly-acrylic acid and esters or -amides thereof,poly-vinylalcohol und esters or -ethers thereof, poly-vinylimidazole,poly-vinylpyrrolidon, und natural polymers like chitosan.

Nonlimiting examples of excipients useful in a composition describedherein include phosphates such as dicalcium phosphate. Nonlimitingexamples of lubricants use in a composition described herein includenatural or synthetic oils, fats, waxes, or fatty acid salts such asmagnesium stearate.

Surfactants for use in a composition described herein can be anionic,anionic, amphoteric or neutral. Nonlimiting examples of surfactantsuseful in a composition described herein include lecithin,phospholipids, octyl sulfate, decyl sulfate, dodecyl sulfate, tetradecylsulfate, hexadecyl sulfate and octadecyl sulfate, Na oleate or Nacaprate, 1-acylaminoethane-2-sulfonic acids, such as1-octanoylaminoethane-2-sulfonic acid, 1-decanoylaminoethane-2-sulfonicacid, 1-dodecanoylaminoethane-2-sulfonic acid,1-tetradecanoylaminoethane-2-sulfonic acid,1-hexadecanoylaminoethane-2-sulfonic acid, and1-octadecanoylaminoethane-2-sulfonic acid, and taurocholic acid andtaurodeoxycholic acid, bile acids and their salts, such as cholic acid,deoxycholic acid and sodium glycocholates, sodium caprate or sodiumlaurate, sodium oleate, sodium lauryl sulphate, sodium cetyl sulphate,sulfated castor oil and sodium dioctylsulfosuccinate,cocamidopropylbetaine and laurylbetaine, fatty alcohols, cholesterols,glycerol mono- or -distearate, glycerol mono- or -dioleate and glycerolmono- or -dipalmitate, and polyoxyethylene stearate.

Nonlimiting examples of sweetening agents useful in a compositiondescribed herein include sucrose, fructose, lactose or aspartame.Nonlimiting examples of flavoring agents for use in a compositiondescribed herein include peppermint, oil of wintergreen or fruit flavorssuch as cherry or orange flavor. Nonlimiting examples of coatingmaterials for use in a composition described herein include gelatin,wax, shellac, sugar or other biological degradable polymers. Nonlimitingexamples of preservatives for use in a composition described hereininclude methyl or propylparabens, sorbic acid, chlorobutanol, phenol andthimerosal.

The hydrate form D described herein may also be formulated aseffervescent tablet or powder, which disintegrate in an aqueousenvironment to provide a drinking solution. A syrup or elixir maycontain the polymorph described herein, sucrose or fructose assweetening agent a preservative like methylparaben, a dye and aflavoring agent.

Slow release formulations may also be prepared from the polymorphdescribed herein in order to achieve a controlled release of the activeagent in contact with the body fluids in the gastro intestinal tract,and to provide a substantial constant and effective level of the activeagent in the blood plasma. The crystal form may be embedded for thispurpose in a polymer matrix of a biological degradable polymer, awater-soluble polymer or a mixture of both, and optionally suitablesurfactants. Embedding can mean in this context the incorporation ofmicro-particles in a matrix of polymers. Controlled release formulationsare also obtained through encapsulation of dispersed micro-particles oremulsified micro-droplets via known dispersion or emulsion coatingtechnologies.

While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art.Typical dosages of the BH4 comprise about 1 to about 20 mg/kg bodyweight per day, which will usually amount to about 5 (1 mg/kg×5 kg bodyweight) to 3000 mg/day (30 mg/kg×100 kg body weight). Such a dose may beadministered in a single dose or it may be divided into multiple doses.While continuous, daily administration is contemplated, it may bedesirable to ceases the BH4 therapy when the symptoms of Phe levels arereduced to below a certain threshold level. Of course, the therapy maybe reinitiated in the event that Phe levels rise again.

It is understood that the suitable dose of a composition according tothe present invention will depend upon the age, health and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired (i.e., the amount of decrease inplasma Phe concentration desired). The frequency of dosing also isdependent on pharmacodynamic effects on Phe levels. If the effect lastsfor 24 hours from a single dose. However, the most preferred dosage canbe tailored to the individual subject, as is understood and determinableby one of skill in the art, without undue experimentation. Thistypically involves adjustment of a standard dose, e.g., reduction of thedose if the patient has a low body weight.

As discussed above, the total dose required for each treatment may beadministered in multiple doses or in a single dose. The BH4 and theprotein compositions may be administered alone or in conjunction withother therapeutics directed to the disease or directed to other symptomsthereof.

As is apparent from the disclosure presented herein, in a broad aspectthe present application contemplates clinical application of acombination therapy comprising a first composition that contains acrystallized BH4 formulation, and a second composition that contains amedical protein formulation (e.g., PHENEX or the like). Therefore, thecompositions should be formulated into suitable pharmaceuticalcompositions, i.e., in a form appropriate for in vivo applications insuch combination therapies. Generally, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals. Preferably, thecrystallized BH4 composition may be such that it can be added directlyto the preexisting protein formulations used for the treatment of PKU.

One will generally desire to employ appropriate salts and buffers torender the BH4 suitable for uptake. Aqueous compositions of the presentinvention comprise an effective amount of the BH4 dissolved or dispersedin a pharmaceutically acceptable carrier or aqueous medium. Suchcompositions may be administered orally or via injection.

The phrase “pharmaceutically or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the therapeutic compositions, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions. In exemplaryembodiments, the medical protein formulation may comprise corn syrupsolids, high-oleic safflower oil, coconut oil, soy oil, L-leucine,calcium phosphate tribasic, L-tyrosine, L-proline, L-lysine acetate,DATEM (an emulsifier), L-glutamine, L-valine, potassium phosphatedibasic, L-isoleucine, L-arginine, L-alanine, glycine, L-asparaginemonohydrate, L-serine, potassium citrate, L-threonine, sodium citrate,magnesium chloride, L-histidine, L-methionine, ascorbic acid, calciumcarbonate, L-glutamic acid, L-cystine dihydrochloride, L-tryptophan,L-aspartic acid, choline chloride, taurine, m-inositol, ferrous sulfate,ascorbyl palmitate, zinc sulfate, L-carnitine, alpha-tocopheryl acetate,sodium chloride, niacinamide, mixed tocopherols, calcium pantothenate,cupric sulfate, thiamine chloride hydrochloride, vitamin A palmitate,manganese sulfate, riboflavin, pyridoxine hydrochloride, folic acid,beta-carotene, potassium iodide, phylloquinone, biotin, sodium selenate,chromium chloride, sodium molybdate, vitamin D3 and cyanocobalamin. Theamino acids, minerals and vitamins in the supplement should be providedin amounts that provide the recommended daily doses of each of thecomponents.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

The active compositions of the present invention include classicpharmaceutical preparations of BH4 which have been discussed herein aswell as those known to those of skill in the art. Protein formulas, suchas, e.g., Phenex, also are known to those of skill in the art.Administration of these compositions according to the present inventionwill be via any common route for dietary supplementation. The protein ispreferably administered orally, as is the BH4.

The active compounds may be prepared for administration as solutions offree base or pharmacologically acceptable salts in water suitably mixedwith a surfactant, such as hydroxypropylcellulose. Dispersions also canbe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The BH4 compositions may be prepared as pharmaceutical forms suitablefor injectable use. Such compositions include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating, such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial an antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The BH4 used in a composition described herein is preferably formulatedas a dihydrochloride salt, however, it is contemplated that other saltforms of BH4 posses the desired biological activity, and consequently,other salt forms of BH4 can be used.

Compositions and methods for producing a stabilized tablet formulationare also disclosed in co-pending U.S. provisional application No.60/629,189 [Attorney Docket No. 30610/40679 filed Nov. 17, 2004, theentirety of which is hereby incorporated by reference.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metals or organicamines.

Pharmaceutically acceptable salts of compounds may also be prepared witha pharmaceutically acceptable cation. Suitable pharmaceuticallyacceptable cations are well known to those skilled in the art andinclude alkaline, alkaline earth, ammonium and quaternary ammoniumcations. Carbonates or hydrogen carbonates are also possible. Examplesof metals used as cations are sodium, potassium, magnesium, ammonium,calcium, or ferric, and the like. Examples of suitable amines includeisopropylamine, trimethylamine, histidine, N,N′ dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N methylglucamine, and procaine.

Pharmaceutically acceptable acid addition salts include inorganic ororganic acid salts. Examples of suitable acid salts include thehydrochlorides, acetates, citrates, salicylates, nitrates, phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include, for example, acetic, citric, oxalic,tartaric, or mandelic acids, hydrochloric acid, hydrobromic acid,sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic,sulfo or phospho acids or N substituted sulfamic acids, for exampleacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid,glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, salicylic acid, 4 aminosalicylic acid, 2 phenoxybenzoic acid, 2acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid;and with amino acids, such as the 20 alpha amino acids involved in thesynthesis of proteins in nature, for example glutamic acid or asparticacid, and also with phenylacetic acid, methanesulfonic acid,ethanesulfonic acid, 2 hydroxyethanesulfonic acid, ethane 1,2 disulfonicacid, benzenesulfonic acid, 4 methylbenzenesulfoc acid, naphthalene 2sulfonic acid, naphthalene 1,5 disulfonic acid, 2 or 3 phosphoglycerate,glucose 6 phosphate, N cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid.

Specifically, BH4 salts with inorganic or organic acids are preferred.Nonlimiting examples of alternative BH4 salts forms includes BH4 saltsof acetic acid, citric acid, oxalic acid, tartaric acid, fumaric acid,and mandelic acid.

The frequency of BH4 dosing will depend on the pharmacokineticparameters of the agent and the routes of administration. The optimalpharmaceutical formulation will be determined by one of skill in the artdepending on the route of administration and the desired dosage. See forexample Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ.Co, Easton Pa. 18042) pp 1435 1712, incorporated herein by reference.Such formulations may influence the physical state, stability, rate ofin vivo release and rate of in vivo clearance of the administeredagents. Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein as well as thepharmacokinetic data observed in animals or human clinical trials.

Appropriate dosages may be ascertained through the use of establishedassays for determining blood levels of Phe in conjunction with relevantdose response data. The final dosage regimen will be determined by theattending physician, considering factors which modify the action ofdrugs, e.g., the drug's specific activity, severity of the damage andthe responsiveness of the patient, the age, condition, body weight, sexand diet of the patient, the severity of any infection, time ofadministration and other clinical factors. As studies are conducted,further information will emerge regarding appropriate dosage levels andduration of treatment for specific diseases and conditions.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in fields of humanmedicine and veterinary medicine. Thus the subject to be treated may bea mammal, preferably human or other animal. For veterinary purposes,subjects include for example, farm animals including cows, sheep, pigs,horses and goats, companion animals such as dogs and cats, exotic and/orzoo animals, laboratory animals including mice rats, rabbits, guineapigs and hamsters; and poultry such as chickens, turkey ducks and geese.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Clinical Evaluation with 6R-Tetrahydrobiopterin

The following example provides guidance on the parameters to be used forthe clinical evaluation BH4 in the therapeutic methods of the presentinvention. As discussed herein throughout, BH4 will be used in thetreatment of HPA including HPA, mild phenylketonuria (PKU) and classicPKU. Clinical trials will be conducted which will provide an assessmentof daily oral doses of BH4 for safety, pharmacokinetics, and initialresponse of both surrogate and defined clinical endpoints. The trialwill be conducted for a minimum, but not necessarily limited to, 6 weeksto collect sufficient safety information for 30 evaluable patients.

The initial dose for the trials will vary from about 10 to about 20mg/kg. In the event that this dose does not produce a reduction inexcess plasma phenylalanine (Phe) levels in a patient, or produce asignificant direct clinical benefit measured as an ability to increasedaily oral Phe intake without increases in plasma Phe levels, the doseshould be increased as necessary, and maintained for an additionalminimal period of, but necessarily limited to, 6 weeks to establishsafety and to evaluate further efficacy. Lower doses, e.g., doses ofbetween 5 to 10 mg/kg also are contemplated.

Measurements of safety will include adverse events, allergic reactions,complete clinical chemistry panel (kidney and liver function),urinalysis, and CBC with differential. In addition, other parametersincluding the reduction in levels of blood Phe levels,neuropsychological and cognitive testing, and global assessments alsowill be monitored. The present example also contemplates thedetermination of pharmacokinetic parameters of the drug in thecirculation, and general distribution and half-life of 6R—BH4 in blood.It is anticipated that these measures will help relate dose to clinicalresponse.

Methods

Patients who have elevated levels of plasma Phe will undergo a baselinea medical history and physical exam, neuropsychological and cognitivetesting, a standard set of clinical laboratory tests (CBC, Panel 20,CH50, UA), levels of urinary pterins, dihydropteridine reductase (DHPR)levels, and a fasting blood (plasma) panel of serum amino acids. Theproposed human dose of 10 to about 20 mg/kg BH4 will be administereddivided in one to three daily doses. The patient will be followedclosely with weekly visits to the clinic. Patients will return to theclinic for a complete evaluation one week after completing the treatmentperiod. Should dose escalation be required, the patients will follow thesame schedule outlined above. Safety will be monitored throughout thetrial.

Enrolled patients will be randomized to receive BH4 or a placebo. Afteran initial two to four-week period all study participants will be placedon a controlled diet with a limited Phe intake for a total of four tosix weeks. After completing the first two to four weeks on dietaryrestriction, all study participants will be crossed-over in theirrandomization and will followed for an additional two to four weeks. Pheblood levels and other biochemical parameters will be followed closelyat the end of each period. Evaluation of neuropsychological outcomeswill include measurements of sustained attention; working memory; andability to perform complex operations. Patients who complete the trial,and who benefited from therapy by showing a beneficial decrease plasmaPhe levels, will be offered continued BH4 therapy thorough an extendedprotocol for as long as safety and efficacy conditions warrant it, oruntil BLA approval.

Diagnosis and Inclusion/Exclusion Criteria

The patient may be male or female, aged twelve years or older with adocumented diagnosis of HPA or mild PKU confirmed by genetic testing andevidence of elevated Phe levels in blood. The study will include HPA orPKU patients who do not accurately follow dietary control. Femalepatients of childbearing potential must have a negative pregnancy test(urine β-hCG) just prior to each dosing and must be advised to use amedically accepted method of contraception throughout the study. Apatient will be excluded from this study if the patient has evidence ofa primary BH4 deficiency, has previously received multiple doses of BH4for more than 1 week of treatment; is pregnant or lactating; hasreceived an investigational drug within 30 days prior to studyenrollment; or has a medical condition, serious intercurrent illness, orother extenuating circumstance that may significantly decrease studycompliance.

Dose, Route and Regimen

Patients will receive BH4 at a dose of 5-10 mg/kg per day. In the eventthat Phe blood levels are not decreased by a reasonable amount and noclinical benefit is observed, the dose will be increased as necessary.Dose escalation will occur only after all patients have undergone atleast 2 weeks of therapy. The daily BH4 dosage will be administeredorally as liquid, powder, tablets or capsules. The total daily dose maybe given as a single dose or perhaps divided in two or three dailydoses. The patients will be monitored clinically as well as for anyadverse reactions. If any unusual symptoms are observed, study drugadministration will be stopped immediately, and a decision will be madeabout study continuation.

Dietary Intervention

Following the initial randomization and two-week treatment period, allstudy participants will undergo dietary counseling and will follow astandard Phe-restricted diet complemented with Phe-specific medicalfoods for a total of four to six weeks. Diets will be managed at homeand dietary intake will be recorded in daily logs. Analyses of theintakes of nutrients and medical foods and the percent of RecommendedDietary Intakes (RDI) will be compared among the treatment groups.

BH4 Safety

BH4 therapy will be determined to be safe if no significant acute orchronic drug reactions occur during the course of the study. Thelonger-term administration of the drug will be determined to be safe ifno significant abnormalities are observed in the clinical examinations,clinical labs, or other appropriate studies.

Example 2 Preparation of Stabilized Crystallized Form of BH4

U.S. Provisional Patent Application Ser. No. 60/520,377, entitled“Polymorphs of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride” filedon Nov. 17, 2003 in the name of Applicants Rudolf MOSER, ofSchaffhausen, Switzerland and Viola GROEHN of Dachsen, Switzerland andassigned Merck-Eprova internal reference number 216, and U.S. patentapplication Ser. No. 10/990,316, entitled “Polymorphs of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride” filed concurrentlyherewith on Nov. 17, 2004 in the name of Applicants Rudolf MOSER, ofSchaffhausen, Switzerland and Viola GROEHN of Dachsen, Switzerland andassigned Merck-Eprova internal reference number 216/US OP (both of theMoser et al. applications are collectively referred to herein as the“Moser Applications” and both are incorporated herein by reference intheir entireties. The examples of that specification describe X ray andRaman spectra studies to characterize the polymorphs of BH4. Each of theBH4 compositions of that application may be used in the treatmentmethods described herein. The following description provides additionalbackground and a brief characterization of some of those exemplarycompositions.

Results obtained during development of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride (see the MoserApplications) indicated that the compound may possess polymorphic forms.The continued interest in this area requires an efficient and reliablemethod for the preparation of individual polymorphs of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride and controlledcrystallization conditions to provide polymorphs, which are preferablystable and easily to handle and to process in the manufacture andpreparation of formulations.

Crystallization techniques well known in the art for producing drugcrystals are used to prepare the prepare the polymorph forms. Suchtechniques include, but are not limited to, techniques such assuspension, precipitation, re-crystallization, evaporation, solvent likewater sorption methods or decomposition of solvates. Diluted, saturatedor super-saturated solutions of the BH4 may be used for crystallization,with or without seeding with suitable nucleating agents. Temperatures upto 150° C. may be applied to form solutions of the drug. Cooling toinitiate crystallization and precipitation down to −100° C. andpreferably down to −30° C. may be applied. Metastable polymorph orpseudo-polymorph forms can be used to prepare solutions or suspensionsfor the preparation of more stable forms and to achieve higherconcentrations in the solutions.

As discussed in the Moser Applications, the polymorph form may beobtained by crystallization of the BH4 from polar solvent mixtures. TheMoser

Applications also describes a process for the preparation of polymorphform of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride, comprisingdissolution, optionally at elevated temperatures, of a solid lowerenergy form than the claimed form of (6R)-L-erythro-tetrahydrobiopterindihydrochloride in a polar solvent mixture, addition of seeds to thesolution, cooling the obtained suspension and isolation of the formedcrystals.

Dissolution may be carried out at room temperature or up to 70° C., Morepreferably the dissolution is carried out at temperatures up to 50° C.The starting material may be added to the final solvent mixture fordissolution, or alternatively the starting material first may bedissolved in water and other solvents may than be added both or oneafter the other solvent. The solution of the BH4 is preferably stirred.Cooling may mean temperatures down to −80° C., preferably down to −40°C. to 0° C. In some embodiments, in order to initiate thecrystallization of the BH4 polymorph, the solution may be seeded.Suitable seeds may include a portion of the polymorph form from anotherbatch of crystals, or crystals having a similar or identical morphology.After isolation, the crystalline form can be washed with acetone ortetrahydrofurane and dried using techniques commonly used for dryingdrug crystals.

The polymorph forms of BH4 described in the Moser Applications are avery stable crystalline form of the drug. The polymorph form can beeasily filtered off, dried and ground to particle sizes desired forpharmaceutical formulations. These outstanding properties renders thispolymorph form especially feasible for pharmaceutical application. Thestability of the polymorph form of BH4 was determined after the BH4×2HCl(the polymorph form) had been stored for 8 months in a minigrip bag at40° C. and 75% relative humidity. Quality was checked in differentintervals throughout the 8 month period by HPLC. After 8 months, thequality and stability of the polymorph was surprisingly similar to thestability seen at time zero:

0 months (at the after 1 after 3 after 8 beginning) after 1 week monthmonths months HPLC 98.4 99.4 98.3 99.1 98.1 [% area]

Accordingly, the Moser Applications provides descriptions of apharmaceutical compositions comprising a polymorph form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride and apharmaceutically acceptable carrier or diluent. Such compositions willbe useful in the therapeutic methods described herein.

In addition to the Moser Applications, those of skill in the art alsoare referred to U.S. Pat. Nos. 6,596,721; 6,441,168; and 6271,374 whichdescribe various methods and compositions for producing stablecrystalline salts of 5-methyltetrahydrofolic acid and methods andcompositions for producing stable forms of 6R tetrandrofolic acid andmethods and compositions for producing stable forms of 6S and 6Rtetrandrofolic acid. Each of these patents are incorporated herein byreference in their entirety as generally teaching methods of producingcrystalline forms of agents and techniques for characterizing suchagents. Such methods may be used in producing stable forms of BH4 foruse as pharmaceutical compositions in the treatment methods taughtherein.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Example 3 Administration of Tetrahydrobiopterin to Humans with ElevatedSerum Phe Levels

An open label, single and multiple dose study was conducted in a totalof 20 patients to demonstrate the safety and efficacy oftetrahydrobiopterin in humans with elevated blood levels ofphenylalanine (>600 μmol/L). Criteria for inclusion in the studyincluded (1) baseline blood Phe levels of >600 μmol/L, (2) age of atleast 8 years. Criteria for exclusion from the study included (1)pregnancy or breastfeeding, (2) concurrent diseases or conditions thatrequire medication or treatment, (3) concurrent treatment with any drugknown to inhibit folate synthesis, and (4) treatment with anyinvestigational drug within 30 days. Each of the patients also wasidentified as having a mutation in the phenylalanine hydroxylase (PAH)gene. Study subjects underwent baseline assessments, including medicalhistory with assessment of phenylketonuria (PKU) orhyperphenylalaninemia (HPA) related signs and symptoms, physicalexaminations, vital signs, serum amino acid (i.e., phenylalanine,tyrosine, and tryptophan) blood levels, and routine laboratory tests(chemistry, hematology, and urinalysis) before inclusion in the study.

The drug tested was (6R)-5,6,7,8-tetrahydrobiopterin, also known as2-amino-6-(1,2-dihydroxypropyl)-5,6,7,8-tetrahydro-3H-pteridin-4-onetetrahydrobiopterin, or sapropterin (BH4 or 6R—BH4. The drug wasobtained in 10 mg or 50 mg oral tablets from Schircks Laboratories,Switzerland (product no. 11.212 (6R)-5,6,7,8-Tetrahydro-L-biopterindihydrochloride). The half-life of the Schircks 6R—BH4 dihydrochloridesalt is approximately 3.5 hours.

Drugs known to inhibit folate synthesis such as bactrim, methotrexate,or 5-FU were not permitted to be administered during the study. Beforeinitiation of 6R—BH4 dosing, a 7 day washout period was required for anydrugs known to inhibit folate synthesis. No investigational drugs werepermitted to be taken during study participation or within 30 days priorto study enrollment.

Within a maximum of 4 weeks following the completion of baselineassessments, eligible subjects began the first stage of the study.Single ascending doses of 10 mg/kg, 20 mg/kg and 40 mg/kg of 6R—BH4 wereadministered orally, with a washout period of at least 7 days betweeneach dose, and subjects were monitored 24 hours after each dose.Subjects underwent a safety assessment and blood amino acid (i.e,phenylalanine, tyrosine, and tryptophan) level measurements before and24 hours after each 6R—BH4 dose. Blood pressure was measured 30 minutesand 1 hour after each dose. Safety assessments included physicalexaminations, vital signs, serial assessment of PKU or HPA related signsand symptoms, recording of adverse events, and monitoring of changes inlaboratory parameters (chemistry, hematology, and urinalysis). Subjectswere instructed to continue their usual diet without any modification,and to record daily intake of food and beverages throughout the study.

After the first stage of the study was completed, subjects entered thesecond stage of the study, during which they received 10 mg/kg of 6R—BH4daily in an oral dosage form, for a total of 7 days. After a washoutperiod of at least 7 days, each subject received 20 mg/kg of 6R—BH4daily for a total of 7 days. During the second stage of the study,subjects were monitored before dosing, at 24 and 72 hours after firstdose, and on the 7th day of dosing at each of the two dose levels.Monitoring included a safety assessment as described above, measurementof serum blood amino acid (i.e, phenylalanine, tyrosine, and tryptophan)levels and evaluation of phenylalanine and tyrosine oral intake.Subjects were instructed to continue their usual diet without anymodification, and to record daily intake of food and beveragesthroughout the study.

After a single dose of 6R—BH4 (10 mg/kg), blood Phe declined 10%±0.26%from baseline. Single doses of 6R—BH4 at 20 mg/kg and 40 mg/kg showedmean declines of 17%±0.28% and 27%±0.25% respectively. The reduction inblood Phe levels appeared to be dose dependent.

FIG. 16 shows mean blood phenylalanine level after 10 and 20 mg/kg6R—BH4 daily for 7 days, in the 14 of 20 patients who responded totreatment. For the purposes of this study, a decline in blood Phe levelsof 30% was considered to be “responsive”, although patients who exhibitless of a decline would still benefit from BH4 treatment. The seven-daytrial showed a sustained decrease in blood Phe concentration in 70% ofthe patients (14/20) taking 20 mg/kg. Of those 14 patients, 10 (71%)responded favorably to 10 mg/kg/day. Blood tyrosine was observed toincrease in some but not all patients; some patients had increasesof >80% from baseline tyrosine levels. The individual blood Pheresponses to multiple doses of 10 mg/kg BH4 are shown in 11 adults (FIG.17) and 9 children (FIG. 19). The individual blood Phe responses tomultiple doses of 20 mg/kg BH4 are shown in 11 adults (FIG. 18) and in 9children (FIG. 20).

Thus, a single-dose loading test was inadequate to identify patients whoresponded to BH4 treatment with a reduction in blood Phe level of 30% ormore. A 7-day loading test successfully identified a high percentage ofresponsive patients. The 20 mg/kg, 7-day loading test with 6R—BH4identified 70% of the PKU patients that responded to 20 mg/kg of BH4. Ofthe 14 responders, 71% also showed a 30% or greater reduction in bloodPhe level with the lower dose of 10 mg/kg 6R—BH4.

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

What is claimed is:
 1. A method for treating classic severephenylketonuria (PKU) in a subject comprising administering to saidsubject a protein-restricted diet in combination with a compositioncomprising tetrahydrobiopterin (BH4) or a precursor or derivativethereof, wherein the combined administration of the protein-restricteddiet and BH4 is effective to lower the phenylalanine concentration inthe plasma of said subject as compared to said concentration in theabsence of said combined administration.
 2. The method of claim 1,wherein said subject does not have a deficiency in BH4 homeostasis. 3.The method of claim 1, wherein said subject has a plasma phenylalanineconcentration of greater than 1000 μM in the absence of a therapeuticregimen.
 4. The method of claim 3, wherein said combined administrationdecreases the plasma phenylalanine concentration of said subject to lessthan 600 μM.
 5. The method of claim 3, wherein said combinedadministration decreases the plasma phenylalanine concentration of saidsubject to less than 500 μM.
 6. The method of claim 3, wherein saidcombined administration decreases the plasma phenylalanine concentrationof said subject to 360 μM±15 μM.
 7. The method of claim 1, wherein saidBH4 is administered in an amount of between about 1 mg/kg to about 30mg/kg.
 8. The method of claim 7, wherein said BH4 is administered in anamount of between about 5 mg/kg to about 30 mg/kg.
 9. The method ofclaim 7, wherein said BH4 is administered in a single daily dose. 10.The method of claim 7, wherein said BH4 is administered in multipledoses on a daily basis.
 11. The method of claim 1, wherein said BH4 isadministered on a daily basis until the plasma phenylalanineconcentration of said subject is decreased to less than 360 μM.
 12. Themethod of claim 11, wherein the plasma phenylalanine concentration ofsaid subject is monitored on a daily basis and said BH4 is administeredwhen a 10% increase in plasma phenylalanine concentration is observed.13. The method of claim 1, wherein said BH4 is administered as astabilized crystallized form.
 14. The method of claim 9, wherein saidstabilized crystallized form of BH4 comprises at least 99.5% pure 6RBH4.
 15. The method of claim 1, wherein said BH4 precursor isdihydrobiopterin (BH2).
 16. The method of claim 1, wherein said BH4precursor is sepiapterin.
 17. The method of claim 1, wherein saidprotein-restricted diet is a phenylalanine-restricted diet wherein thetotal phenylalanine is restricted to less than 600 mg per day.
 18. Themethod of claim 1, wherein said protein-restricted diet is aphenylalanine-restricted diet wherein the total phenylalanine isrestricted to less than 300 mg per day.
 19. The method of claim 1,wherein said protein-restricted diet is supplemented with tyrosine. 20.The method of claim 1, wherein said protein-restricted diet comprises ahigh content of one or more amino acids selected from valine, isoleucineand leucine.
 21. The method of claim 1, wherein said BH4 is administeredorally.
 22. The method of claim 1, wherein said subject has beendiagnosed as having a mutant phenylalanine hydroxylase (PAH).
 23. Themethod of claim 22, wherein said mutant PAH comprises a mutation in thecatalytic domain of PAH.
 24. The method of claim 22, wherein saidmutation comprises one or more mutations selected from the groupconsisting of F39L, L48S, I65T, R68S, A104D, S110C, D129G, E178G, V190A,P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G,A395P, P407S, and Y414C.
 25. The method of claim 1, wherein said subjectis subject does not manifest symptoms of L-dopa neurotransmitterdeficiency.
 26. The method of claim 1, wherein said protein-restricteddiet comprises a protein supplement and said BH4 is provided in the samecomposition as said protein supplement.
 27. A method for the treating apregnant female having hyperphenylalaninemia (HPA) comprisingadministering to said subject a protein-restricted diet in combinationwith a composition comprising tetrahydrobiopterin (BH4) or a precursoror derivative thereof, wherein the combined administration of theprotein-restricted diet and BH4 is effective to lower the phenylalanineconcentration in the plasma of said subject as compared to saidconcentration in the absence of said combined administration.
 28. Themethod of claim 27, wherein said subject has a plasma phenylalanineconcentration of greater than 180 μM but less than 600 μM.
 29. Themethod of claim 27, wherein said subject has a plasma phenylalanineconcentration of greater than 600 μM but less than 1200 μM.
 30. Themethod of claim 27, wherein said subject has a plasma phenylalanineconcentration of greater than 1200 μM.
 31. The method of any of claims 1to 30, wherein said composition further comprises a folate.
 32. Themethod of claim 31, wherein said folate comprises a tetrahydrofolateselected from the group consisting of tetrahydrofolate is5-formyl-(6S)-tetrahydrofolic acid and salts thereof,5-methyl-(6S)-tetrahydrofolic acid and salts thereof,5,10-methylene-(6R)-tetrahydrofolic acid and salts thereof,5,10-methenyl-(6R)-tetrahydrofolic acid and salts thereof,10-formyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolicacid salts thereof, (6S)-tetrahydrofolic acid and salts thereof, andcombinations of the foregoing.
 33. The method of claim 31, wherein saidcomposition further comprises arginine.
 34. A method of treating apatient having above normal concentration of plasma phenylalaninecomprising administering to said patient a stabilized BH4 composition inan amount effective to produce a decrease in the plasma phenylalanineconcentration of said patient.
 35. The method of claim 34, wherein saidstabilized BH4 composition is stable at room temperature for more than 8hours.
 36. The method of claim 34, wherein said patient has a plasmaphenylalanine concentration greater than 180 μM prior to administrationof said BH4.
 37. The method of claim 34, wherein said patient has aplasma phenylalanine concentration of between 180 μM and 360 μM.
 38. Themethod of claim 34, wherein said patient has a plasma phenylalanineconcentration of between 200 μM and 600 μM.
 39. The method of claim 34,wherein said patient has a plasma phenylalanine concentration of between600 μM and 1200 μM.
 40. The method of claim 34, wherein said patient hasa plasma phenylalanine concentration of between 600 μM and 1200 μM. 41.The method of claim 34, wherein said patient has a plasma phenylalanineconcentration greater than 1200 μM.
 42. The method of claim 34, whereinsaid patient is an infant.
 43. The method of claim 42, wherein saidinfant has a plasma phenylalanine concentration greater than 1200 μM.44. The method of claim 34, wherein said patient is pregnant.
 45. Themethod of claim 44, wherein said patient has a plasma phenylalanineconcentration of between about 200 μM to about 600 μM.
 46. The method ofclaim 44, wherein said patient has a plasma phenylalanine concentrationgreater than 1200 μM.
 47. The method of claim 34, wherein said patientis a female of child-bearing age that is contemplating pregnancy. 48.The method of claim 47, wherein said patient has a plasma phenylalanineconcentration of between about 200 μM to about 600 μM.
 49. The method ofclaim 47, wherein said patient has a plasma phenylalanine concentrationgreater than 1200 μM, and said method further comprising administering aprotein-restricted diet to said patient.
 50. A method of treating apatient having phenylketonuria, comprising administering to said patienta stabilized BH4 composition in an amount effective to produce adecrease in the plasma phenylalanine concentration of said patientwherein said patient has been diagnosed as unresponsive to a BH4 loadingtest.
 51. The method of claim 50, said method comprising administeringbetween about 10 mg BH4/kg body weight to about 200 mg BH4/kg bodyweight.
 52. The method of claim 50, wherein said BH4 is administered inorally, subcutaneously, sublingually, parenterally, per rectum, per andnares.
 53. The method of claim 50, wherein said BH4 is administereddaily.
 54. The method of claim 50, wherein said BH4 is administered onevery alternative day.
 55. The methods of claim 50, wherein said BH4 isadministered weekly.
 56. The method of claim 50, wherein said BH4 isadministered in combination with a protein-restricted diet.
 57. Themethod of claim 50, wherein said BH4 is administered as part of acomponent of a therapeutic protein formulation.
 58. The method of claim56, wherein said protein-restricted diet comprises a normal diet oflow-protein containing foodstuff.
 59. The method of claim 56, whereinsaid protein-restricted diet comprises a phenylalanine-free proteindiet.
 60. The methods of claim 56, wherein said protein-restricted dietis supplemented with non-phenylalanine containing protein supplements.61. The method of claim 60, wherein said non-phenylalanine containingprotein supplements comprise tyrosine.
 62. A method of treating aninfant having phenylketonuria, comprising administering a stabilized BH4composition to said patient in an amount effective to produce a decreasein the plasma phenylalanine concentration of said infant wherein saidinfant is between 0 and 3 years of age and said infant has a plasmaphenylalanine concentration of between about 360 μM to about 4800 μM.63. The method of claim 62, wherein prior to said administering of BH4said infant has a phenylalanine concentration of about 1200 μM and saidadministering of BH4 decreases said plasma phenylalanine concentrationto about 1000 μM.
 64. The method of claim 63, wherein prior to saidadministering of BH4 said infant has a phenylalanine concentration ofabout 800 μM and said administering of BH4 decreases said plasmaphenylalanine concentration to about 600 μM.
 65. The method of claim 63,wherein prior to said administering of BH4 said infant has aphenylalanine concentration of about 400 μM and said administering ofBH4 decreases said plasma phenylalanine concentration to about 300 μM.66. The method of claim 63, wherein said administering of BH4 decreasessaid plasma phenylalanine concentration 360±15 μM.
 67. The method of anyof claims 34 to 66, further comprising administering a folate.
 68. Themethod of claim 67, wherein said folate comprises a tetrahydrofolateselected from the group consisting of tetrahydrofolate is5-formyl-(6S)-tetrahydrofolic acid and salts thereof,5-methyl-(6S)-tetrahydrofolic acid and salts thereof,5,10-methylene-(6R)-tetrahydrofolic acid and salts thereof,5,10-methenyl-(6R)-tetrahydrofolic acid and salts thereof,10-formyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolicacid salts thereof, (6S)-tetrahydrofolic acid and salts thereof, andcombinations of the foregoing.
 69. The method of claim 68, furthercomprising administering arginine.
 70. A composition comprising astabilized, crystallize form of BH4 that is stable at room temperaturefor more than 8 hours and a pharmaceutically acceptable carrier, diluentor excipient.
 71. The composition of claim 70, wherein said compositionfurther comprises a medical protein supplement.
 72. The composition ofclaim 70, wherein said BH4 composition is part of an infant formula. 73.The composition of claim 71, wherein said protein supplement isphenylalanine free.
 74. The composition of claim 73, wherein saidprotein supplement is fortified with L-tyrosine, L-glutamine,L-carnitine at a concentration of 20 mg/100 g supplement, L-taurine at aconcentration of 40 mg/100 g supplement and selenium.
 75. Thecomposition of claim 74, wherein said protein supplement furthercomprises the recommended daily dose of calcium and phosphorus.
 76. Thecomposition of claim 74, wherein said protein supplement furthercomprises the recommended daily dose of one or more amino acids selectedfrom the group consisting of L-leucine, L-proline, L-lysine acetate,L-valine, L-isoleucine, L-arginine, L-alanine, glycine, L-asparaginemonohydrate, L-tryptophan, L-serine, L-threonine, L-histidine,L-methionine, L-glutamic acid, and L-aspartic acid.
 77. The compositionof claim 74, wherein said protein supplement is further fortified withthe recommended daily dosage of vitamins A, D and E.
 78. The compositionof claim 73, wherein said protein supplement comprises a fat contentthat provides at least 40% of the energy of said supplement.
 79. Thecomposition of claim 73, wherein said composition is in the form of apowder.
 80. The composition of claim 71, wherein said composition is inthe form of a protein bar.
 81. The composition of claim 70, furthercomprises a folate.
 82. The composition of claim 81, wherein said folatecomprises a tetrahydrofolate selected from the group consisting oftetrahydrofolate is 5-formyl-(6S)-tetrahydrofolic acid and saltsthereof, 5-methyl-(6S)-tetrahydrofolic acid and salts thereof,5,10-methylene-(6R)-tetrahydrofolic acid and salts thereof,5,10-methenyl-(6R)-tetrahydrofolic acid and salts thereof,10-formyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolicacid salts thereof, (6S)-tetrahydrofolic acid and salts thereof, andcombinations of the foregoing.
 83. The composition of claim 82, furthercomprising arginine.